JO - Stony Brook University · 1. EXPLORATION OF ORGANOFLUORINE CHEMISTRY BY MEANS OF TRANSITION-METAL CATALYSIS My ﬁrst encounter with “ﬂuorine chemistry” was in the late

JOC The Journal of Organic Chemistry July 5, 2013 VOluME 78, NuMBER 13 pubs.acs.org/joc

www.acs.org

About the Cover:

Research endeavors are represented on the exploration of organofluorine chemistry at the

multidisciplinary interface of biology and chemistry. The background is the tubulin-bound

“REDOR–Taxol” molecule and a macrocyclic analog mimicking it at the β-tubulin binding site

for Taxol. See Ojima, 6358. Professor Iwao Ojima is the recipient of the 2013 ACS Award for

Creative Work in Fluorine Chemistry. View the article.

http://dx.doi.org.libproxy.cc.stonybrook.edu/10.1021/jo400301u

Exploration of Fluorine Chemistry at the Multidisciplinary Interfaceof Chemistry and BiologyIwao Ojima*

Department of Chemistry and Institute of Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, New York11794-3400, United States

ABSTRACT: Over the last three decades, my engagement in “fluorinechemistry” has evolved substantially because of the multidisciplinary nature ofthe research programs. I began my research career as a synthetic chemist inorganometallic chemistry and homogeneous catalysis directed toward organicsynthesis. Then, I was brought into a very unique world of “fluorine chemistry”in the end of 1970s. I started exploring the interface of fluorine chemistry andtransition metal homogeneous catalysis first, which was followed by aminoacids, peptides, and peptidomimetics for medicinal chemistry. Since then, I havebeen exploring the interfaces of fluorine chemistry and multidisciplinary fieldsof research involving medicinal chemistry, chemical biology, cancer biology, andmolecular imaging. This perspective intends to cover my fruitful endeavor in theexploration of fluorine chemistry at the multidisciplinary interface of chemistryand biology in a chronological order to show the evolution of my researchinterest and strategy.

■ INTRODUCTIONThe extraordinary potential of fluorine-containing biologicallyrelevant molecules in peptide/protein chemistry, medicinalchemistry, chemical biology, pharmacology, drug discovery aswell as diagnostic and therapeutic applications was recognizedby researchers who are not in the traditional fluorine chemistryfield, and thus a new wave of fluorine chemistry has beenrapidly expanding its biomedical frontiers. In fact, theimportance of fluorine in bioorganic and medicinal chemistryhas been demonstrated by a large number of fluorinatedcompounds approved by the FDA for medical use.1−3

According to our survey in 2008, 138 fluorine-containingdrugs have received FDA approval for human diseases (ofwhich 23, however, have been discontinued from the market),while 33 are currently in use for veterinary applications.4 Thesestatistics make fluorine the “second-favorite heteroatom” afternitrogen in drug design.Small atomic radius, high electronegativity, nuclear spin of 1/

2, and low polarizability of the C−F bond are among the specialproperties that render fluorine so attractive. These atomicproperties translate widely into equally appealing attributes offluoroorganic compounds. Higher metabolic stability, oftenincreased binding to target molecules, and increased lip-ophilicity and membrane permeability are some of theproperties associated with the replacement of a C−H or C−O bond with a C−F bond in biologically active compounds.Because of the recognized value of fluorine, it is now a commonpractice in drug discovery to study fluoro-analogues of leadcompounds under development. It should be noted that in2006 the best and the second best selling drugs in the worldwere Lipitor (atorvastatin calcium) (by Pfizer/Astellas; $14.4billion/year) and Advair (USA)/seretide(EU) (a mixture of

fluticasone propionate and salmeterol) (by GlaxoSmithKline;$6.1 billion/year), which contain one and three fluorine atoms,respectively.5 These huge successes of fluorine-containing drugskeep stimulating research on fluorine in medicinal chemistry fordrug discovery. As such, it is not an exaggeration to say thatevery new drug discovery and development today exploresfluorine-containing drug candidates without exception.Although medicinal chemists have been introducing fluorine

into bioactive molecules on the basis of experience andintuition, it is only recently that experimental and computa-tional studies have been conducted to better understand howthe introduction of fluorine into small drug molecules results inhigher binding affinities and selectivity.6 An understanding ofhow the replacement of H with F affects the electronic natureand conformation of small molecules is crucial for predictingthe interaction of fluoroorganic molecules with proteins andenzymes. In addition, 19F NMR has found numerousapplications to molecular imaging and promoted the develop-ment of molecular probes for imaging. The sensitivity of 19FNMR spectroscopy, along with large 19F−1H couplingconstants and the virtual absence of 19F in living tissues,makes incorporation of fluorine into bioactive compounds aparticularly powerful tool for the investigation of biologicalprocesses.7−9 Also, applications of 18F-PET (positron emissiontomography), a powerful in vivo imaging technology inoncology, neurology, psychiatry, cardiology, and other medicalspecialties, have already become an essential part of medicalcare. In addition, 18F-PET has emerged as an important tool in

Received: February 8, 2013Published: April 24, 2013

Perspective

pubs.acs.org/joc

© 2013 American Chemical Society 6358 dx.doi.org/10.1021/jo400301u | J. Org. Chem. 2013, 78, 6358−6383

pubs.acs.org/joc

drug development, especially for accurate measurements ofpharmacokinetics and pharmacodynamics.10

There is a strong demand for developing new and efficientsynthetic methods as well as expanding the availability ofversatile fluorine-containing synthetic building blocks andintermediates to promote medicinal chemistry, chemicalbiology, and molecular imaging research.11−19 The limitedavailability of fluorochemicals for bioorganic and medicinalchemistry as well as pharmaceutical and agrochemicalapplications is mainly due to the exceptional properties andhazardous nature of fluorine and fluorochemical sources. Also,in many cases, synthetic methods developed for ordinaryorganic molecules do not work well for fluorochemicals becauseof their unique reactivity.11−19 Therefore, the new and efficientsynthetic methods applicable to organofluorine compounds,including 18F radiotracers, need to be continuously developed.This Perspective was commissioned on the occasion of my

receiving the 2013 ACS Award for Creative Work in FluorineChemistry. Accordingly, I would like to review the researchstrategy and programs in my laboratory in the last threedecades in perspective, which would be useful to foresee thefuture directions in organofluorine chemistry at the multi-disciplinary interface of chemistry and biology.

1. EXPLORATION OF ORGANOFLUORINE CHEMISTRYBY MEANS OF TRANSITION-METAL CATALYSIS

My first encounter with “fluorine chemistry” was in the late1970s, when I was a Group Leader for organometallicchemistry and homogeneous catalysis as well as organicsynthesis at the Sagami Institute in Japan. At that time, theresearch council of the institute decided to add “fluorinechemistry” to one of its strategic research areas. To me, one ofthe most inspirational reports back then was the boldexperiments done by Leland Clark.20 In this paper, Clarkdemonstrated the capability of perfluorocarbons (PFCs) todeliver oxygen to a living mouse placed deep in a beaker filledwith PFCs,20 which clearly implied the extraordinary nature ofthe world of “fluorine”. Of course, fluoropolymers, especially“Teflons”, fluorosilicones, coolants/refrigerants, extinguishers,aerosols, etc., were the representative fluorochemicals com-monly used in daily life. As for pharmaceutical drugs, only 5-FUwas widely recognized followed by 9α-fluorohydrocortisone atthat time.From a strategic point of view based on synthetic organic

chemistry, I reviewed and critically analyzed potential newfields of research. Then, I found that the synthetic methods bymeans of homogeneous catalysis were virtually nonexistent influorine chemistry. Accordingly, I decided to explore theinterface of traditional fluorine chemistry and transition-metal-catalyzed reactions to establish a new and interdisciplinaryresearch program in my laboratory.We started our research program from the development of

(i) unique hydrocarbonylations of fluoro-olefins that wouldprovide versatile intermediates for the synthesis of a variety oforganofluorine compounds and (ii) one-pot multistepprocesses exploiting the cobalt-catalyzed amidocarbonylationof aldehydes as a key unit reaction since this reaction wouldfurnish important fundamental biochemicals, i.e., N-acyl α-amino acids, from an aldehyde, amide, carbon monoxide, andhydrogen. This research program proceeded smoothly, leadingto successful findings of (i) the unique and remarkable effectsof organofluorine substituents on the regioselectivity in thehydrocarbonylations of fluoro-olefins and the application of the

highly regioselective hydroformylation to the synthesis offluoro-amino acids, (ii) a novel ureidocarbonylation processthat gives 5-(trifluoromethy1)dihydrouracils in one step, and(iii) the hydroformylation−amidocarbonylation of fluoro-olefins catalyzed by Co−Rh mixed-metal systems.Although this program was initiated at the Sagami Institute in

Japan, I moved to the State University of New York at StonyBrook in 1983 and continued the exploration and expansion ofthe scope of the research program.

1.1. Hydroformylation of Fluoro-olefins. Hydroformy-lation of alkenes is important for the practical synthesis ofaldehydes,21 and extensive studies were performed on thedetailed mechanism of the reaction as well as applications toorganic syntheses by the early 1980s.22,23 Little was known,however, about the reactions of alkenes bearing perfluoroalkylor perfluoroaryl substituents when we started our investiga-tion.24 It has been shown that the introduction of atrifluoromethyl or a fluoroaromatic group into organiccompounds often brings about unique chemical and biologicalproperties.25−28 Thus, the development of new syntheticmethods that can introduce these fluoro groups efficientlyand selectively to the desired molecules from readily availablematerials had an obvious significance. In this respect,commercially available fluoro-olefins such as 3,3,3-trifluoropro-pene (TFP), vinyl fluoride (VF), and pentafluorostyrene (PFS)were recognized as very useful starting materials. Thus, westudied the hydroformylation of a variety of fluoro-olefins (eq1) as one of our approaches to the functionalizations of these

building blocks by means of transition-metal catalysts. Then, wefound unusually high regioselectivities and a remarkabledependency of the regioselectivities of the reaction on thecatalyst metal species, which was unique in comparison with thehydroformylation of ordinary alkenes.29−31

1.1.1. Remarkable Dependence of Regioselectivity on theCatalyst Metal Species. The hydroformylation of TFP wascarried out with Co2(CO)8, Ru3(CO)12, Rh6(CO)16, and PtCl,(DIOP)/SnC12, which are typical hydroformylation catalysts,at 100 °C and 100 atm (CO/H2 = 1) for the Co, Pt, and Rucatalysts and at 80 °C and 110 atm (CO/H2 = 1) for the Rhcatalyst (Scheme 1).29−31 The reaction of TFP catalyzed byCo2(CO)8 gave (trifluoromethy)propanals (TFMPAs) in 95%yield, in which a “normal” (or linear) aldehyde,

Scheme 1. Hydroformylation of TFP Catalyzed by Co, Pt,Ru, and Rh complexes

The Journal of Organic Chemistry Perspective

dx.doi.org/10.1021/jo400301u | J. Org. Chem. 2013, 78, 6358−63836359

CF3CH2CH2CHO (3-TFMPA), was formed with highregioselectivity (93%). In sharp contrast with Co2(CO)8, Rh-carbonyl cluster Rh6(CO)16 exhibited extremely high catalyticactivity and regioselectivity (96%) to give “iso” (or branched)aldehyde, CF3(CH3)CHCHO (2-TFMPA). The Pt catalyst,PtCl2(DIOP)/SnCl2, favored the formation of normal aldehyde(n/iso =71/29), while Ru3(CO)12 gave isoaldehyde as the mainproduct (n/iso =15/85). In both cases, a substantial amount ofhydrogenated product, CF3CH2CH3, was formed (25−38%).Addition of PPh3 to the Co, Ru, and Rh catalysts considerablydecreased the catalytic activities but somewhat increased theisoaldehyde selectivity. The result made a sharp contrast to thecases of ordinary olefins, where the addition of PPh3 increasednormal aldehyde se1ectivity.Since Rh6(CO)16 gave excellent regioselectivity for the

formation of 2-TFMPA, several other Rh catalysts wereemployed and their catalytic activities as well as regioselectiv-ities examined. The results clearly indicated that the Rh(I)complexes having chlorine as a ligand, such as RhC1(PPh3)3,were less active than HRh(CO)(PPh3)3, Rh−C, Rh4(CO)12,and Rh6(CO)16, but the regioselectivity was virtually the samein all cases examined. Consequently, it was concluded that thenature of the central metal of the catalyst played a key role indetermining the regioselectivity of the reaction. It wasnoteworthy that the metal species dependency of theregioselectivity in the this reaction was remarkable comparedto that reported for propene.22

The reaction of PFS was carried out in a similar manner at 90°C and 80 atm, using Co, Pt, Ru, and Rh catalysts.29−31

Rhodium catalysts exhibited high catalytic activity to giveisoaldehyde, C6F5(CH3)CHCHO (2-PFPPA), with excellentregioselectivity (97−98%) in quantitative yields, whileCo2(CO)8 gave normal aldehyde (3-PFPPA) as the majorproduct, with regioselectivity (79−90%) not as high as thatobserved in the reaction of TFP. The Ru catalyst, Ru3(CO)12,showed rather low catalytic activity (49% conversion), givingisoaldehyde as the major isomer (22% yield, b/n = 74/26),accompanied by a substantial amount of hydrogenated product,C6F5CH2CH3 (25%). The Pt catalyst, PtCl2(DIOP)/SnCl2,showed a high catalytic activity (100% conversion in 4 h, 76%aldehyde yield), but virtually no regioselectivity was observedand the hydrogenation of PFS took place as a severe sidereaction (20%). Thus, the dependency of regioselectivity on themetal species was similar to that for TFP, and the observedregioselectivity was also remarkably high compared with thatreported for styrene.32−34

A kinetic study was performed for the Rh4(CO)12- andCo2(CO)8-catalyzed reactions of PFS.30 At 100 °C and 82 atm(CO/H2 = 1) with 1.0 × 10−5 M catalyst concentration, theRh-catalyzed reaction was first order in PFS concentration, andthe apparent rate constant was calculated to be 6.2 × 104 s−l;i.e., the turnover number was estimated to be 55800 h−1 per Rhmetal. The Co-catalyzed reaction with 1.0 × 10−2 M catalystconcentration at 100 °C and 82 atm (CO/H2 = 1) was also firstorder in PFS concentration, and the apparent rate constant wascalculated to be 1.6 × 10−5 s−l; i.e., the turnover number percobalt metal was 2.88 h−l. Thus, the Rh catalyst was ca. 20000times more active than the Co catalyst per metal provided thatall metal species participate in the catalysis.On the basis of the fact that the addition or the introduction

of tertiary phosphines to the catalyst caused only a slightchange in regioselectivity, in sharp contrast to the hydro-formylation of propene or styrene using the same catalysts,

both TFP and PFS should have a large binding constant withcatalyst metal species, and thus, these fluoro-olefins should actas important ligands that stabilize the catalysts during thereaction.30

In order to examine the effects of perfluoroalkyl substituentslonger than the trifluoromethyl group on the regioselectivity,the reactions of other fluoro-olefins of the type RfCHCH2catalyzed by Rh4(CO)12 were carried out, wherein Rf were C2F5(PFB), C3F7 (HPFP), and C8F17 (HPDFD) (eq 2).30 The

reactions gave the corresponding branched aldehydes withlower regioselectivity (72−83%) than that for TFP under thestandard conditions, i.e., at 80 °C and 100 atm (CO/H2 = 1).Nevertheless, higher selectivity (91−97%) was achieved whenthe reactions were carried out at 60 °C.The reaction of vinyl fluoride (VF) catalyzed by Rh, Ru, and

Co complexes was also carried out (eq 3), which gave 3-fluoropropanal (2-FPA) exclusively regardless of the catalystspecies used.30

Mechanism of the Highly Regioselective Hydroformyla-tion. The observed marked dependence of regioselectivity onthe catalyst species was accommodated by taking into accountthe stability of isoalkyl−[M] species, the capability of isoalkyl−[M] species for isomerization, and the relative rate of themigratory insertion of CO into isoalkyl−[M] and n-alkyl−[M]bonds.29,30 As shown in Scheme 2, when a substituent bearing astrong “group electronegativity” is introduced into an olefin, themetal−Cα bond of a π-olefin−[M] complex (IA) should bestronger than the metal−Cβ bond because of substantial

Scheme 2. Mechanism of Highly RegioselectiveHydroformylation of Fluoro-olefins



stabilization of the formal negative charge developing on Cα.Thus, the formation of isoalkyl−[M] species (IIiso) should bemuch more favorable than that of n-alkyl−[M] species (IIn)regardless of the group VIII transition-metal species. In fact, theresults of the reactions of vinyl fluoride (VF) provide strongsupporting evidence for this hypothesis.The iso/n ratio of aldehydes should reflect the ratio of the

intermediate iso- and n-acyl−[M] species (IIIiso and IIIn)(Scheme 2) under sufficient pressure of hydrogen. Thus, it isdeduced that in the Rh-catalyzed reaction, kiso ≫ k‑i and kn

CO

≫ k‑n, and thus the initially formed isoalkyl−[Rh] species (IIiso,M = Rh) generates the isoacyl-[Rh] species (IIIiso, M = Rh)and gives the isoaldehyde with high regioselectivity. In sharpcontrast, the rate constants in the Co-catalyzed reaction are k‑n≫ kn

CO and k‑i ≫ kisoCO; kn

CO > kisoCO. This is because the CO

insertion to IIn (M = Co) is sterically less demanding than thatto IIiso (M = Co). Accordingly, the alkyl−[M] intermediates,IIiso and IIn (M = Co), should be in a pre-equilibrium, and thenthe reaction gives the normal aldehyde selectively. The Rh- andCo-catalyzed reactions are extremely selective cases, and the Pt-and Ru-catalyzed reactions are in between the two extremecases.In addition to these kinetic aspects, we should take into

account the fundamental difference between each isoalkyl−[M]intermediate, i.e., the size of the metal and the polarizability ofthe metal−carbon bond. Thus, the relative stability of IIiso canbe estimated to increase in the order Rf(Me)CH-CoLn <Rf(Me)CH-PtLn < Rf(Me)CHRuLn < Rf(Me)CH-RhLn.

30

It is worth noting that these mechanistic details wererevealed because of the use of fluoro-olefins as uniquesubstrates for the hydroformylation reaction. In this case,organometallic chemistry and catalysis research greatlybenefited from fluorine compounds. In turn, fluorine chemistryalso benefited from the discovery of highly regioselectivehydroformylation processes, which provided versatile fluorine-containing aldehydes. In fact, immediately after the discovery ofhighly regioselective hydroformylation of TFP by Rh-catalyzedprocess, I envisioned that we should be able to produce a seriesof “CF3-chemicals” from TFMAs.35

1.2. Hydroesterification and Hydrocarboxylation ofFluoro-olefins. Hydrocarbonylations of olefins serves as aconvenient method for the synthesis of the correspondingesters or carboxylic acids.22,36 Despite extensive mechanisticstudies as well as applications of the reactions to organicsyntheses, little attention had been paid to the reactions offluoro-olefins before we started the investigation on thissubject.The screening of typical transition-metal complexes in the

hydrocarbonylations of TFP and PFS revealed that only Pdcomplexes with phosphine ligands showed sufficient catalyticactivity to promote the reaction under the given reactionconditions.37 As Scheme 3 shows, the Pd-complex-catalyzedhydroesterification of TFP and PFS gave branched esters,Rf(Me)CHCOOR, in good to excellent regioselectivity, whilethe corresponding hydrocarboxylation afforded linear acids,RfCH2CH2COOH, in excellent yield and regioselectivity.Plausible mechanisms were proposed to accommodate theobserved marked difference in regioselectivity for these tworeactions.

2. SYNTHESIS OF FLUORO ANALOGUES OFALIPHATIC AND AROMATIC α-AMINO ACIDS

By the mid 1980s, it was shown that fluorinated analogues ofnaturally occurring biologically active compounds oftenexhibited unique physiological activities.25,28,38 For example,fluorinated pyrimidines acted as anticancer/antiviral agents, andsome fluoro-aromatic compounds as well as CF3-aromaticcompounds were used as nonsteroidal anti-inflammatory drugs,antifungal agents, human antiparasitic agents, central nervoussystem agents for psycho-pharmacology, diuretics agents, andantihypertensive agents. Some fluoro-amino acids acted as“suicide substrate enzyme inactivators”, showing strongantibacterial activities and some of them also acted asantihypertensive agents.28 In the mid to late 1980s, there wasan increasing interest in the incorporation of fluoro-amino acidsinto peptides.39−43 Accordingly, it was timely and important todevelop new and efficient methods for the synthesis of fluoro-amino acids at that time (and even now in the 2010s!).We found that the fluoro-aldehydes obtained in the

hydroformylation of TFP and PFS, described above, served asexcellent intermediates for the synthesis of fluoro-amino acids.Thus, we developed efficient synthetic routes to 4,4,4-trifluorovaline (TFV), 5,5,5-trifluoronorvaline (TFNV), 5,5,5-trifluoroleucine (TFL), 6,6,6-trifluoronorleucine (TFNL),4,5,6,7-tetrafluorotryptophan (tryptophan-f4), and related com-pounds from the fluoro-aldehydes by means of transition-metal-catalyzed transformations as well as enzymatic reactions.44

2.1. 4,4,4-Trifluorovaline (TFV) and 5,5,5-Trifluoronor-valine (TFNV). TFV and TFNV were synthesized using Co-catalyzed amidocarbonylation of 2-TFMPA and 3-TFMPA,respectively. The amidocarbonylation of 2-TFMPA and 3-TFMPA with acetamide catalyzed by Co2(CO)8 (CO/H2 (1/1) 100 atm, 120 °C) gave Ac-TFV and N-Ac-TFNV,respectively, in good yields, which were further hydrolyzed tothe corresponding free amino acids (eqs 4 and 5).44

We also successfully carried out the kinetic optical resolutionof Ac-TFNV, using porcine kidney acylase I (25 °C, pH 7.0) togive (S)-TFNV and (R)-Ac-TFNV with high enantiopurities,which were readily separated (eq 6).44 (R)-Ac-TFNV wasfurther hydrolyzed to (R)-TFNV with 3 M HCl. Theenantiopurities of (S)-TFNV and (R)-TFNV were determinedby Mosher’s MTPA method45 (1H and 19F NMR).

Scheme 3. Pd-Catalyzed Hydroesterification andHydrocarboxylation of TFP and PFS



It has been shown that TFNV inhibits the growth of E. coliand may be used as a growth regulatory factor in micro-biology.46 TFV serves as a modifier of biologically activepeptides in protein engineering and chemical biology. In fact,both TFNV and TFV are commercially available from morethan several vendors now, and indeed, (2S)-TFV has beenextensively used in protein design and chemical biology, as weenvisioned in late 1980s.47

2.2. 5,5,5-Trifluoroleucine (TFL) and 6,6,6-Trifluoro-norleucine (TFNL). TFL and TFNL were synthesized viaazlactones prepared from 2-TFMPA and 3-TFMPA, respec-tively (Scheme 4, (a) and (b)).44 The azlactones were subjectedto alcoholysis to give the corresponding dehydroamino acidesters 3 and 4. (Z)-Dehydroamino acid 3c was obtained byhydrolysis of 1-Z. Then, the (Z)-dehydroamino acid and esters(3-Z and 4-Z) were hydrogenated over Pd/C followed by

hydrolysis to give the corresponding amino acids TFL andTFNL. The azlactones (1-Z and 2-Z) were also treated withhydriodic acid/red phosphorus to give TFL and TFNL directly.It is worthy of note that the chiral 2,2,2-trifluoroisopropyl

group acted as an effective stereogenic center in thehydrogenation of 3-Z over Pd/C, yielding Bz-TFL-OR(Scheme 4, (a)).44 Thus, the hydrogenation of 3a-Z, 3b-Z,and 3c-Z in THF at ambient temperature and pressure of H2gave Bz-TFL-OMe (85:15 dr), Bz-TFL-OEt (90:10 dr), andBz-TFL−OH (82:18 dr), respectively, in quantitative yield. Itwas rather surprising that the “chiral isopropyl group” was ableto induce a high degree of stereoselectivity. It was stronglysuggested that the trifluoromethyl group was not only a bulkiersubstituent than methyl but also imposed a unique electroniceffect on the Pd−metal surface.Optically active (S)-N-Bz-TFNL-OMe (87−89% ee) was

obtained quantitatively by asymmetric hydrogenation of 4a-Z(R = Me) using a cationic Rh catalyst with diPAMP48 at 40 °Cand 1 atm of H2 in ethanol.44 (S)- and (R)-TFNL withexcellent enantiopurities (>98% ee) were obtained throughenzymatic resolution of racemic Ac-TFNL using porcine kidneyacylase I in a manner similar to that for Ac-TFNV (eq 7).44

We envisioned that TFL and TFNL would serves as uniquemodifiers of biologically active peptides in protein engineeringand chemical biology. As we anticipated in the late 1980s, bothTFL and TFNL are commercially available from more thanseveral vendors now, and indeed, (2S)-TFL has beenextensively used in protein design and chemical biology.47

2.3. 4,5,6,7-Tetrafluorotryptophan and Related Com-pounds. As the importance of biologically active compoundsbearing the indole skeleton such as tryptophan, tryptamine,indoleacetic acid, and alkaloids was well recognized, tetrafluoroanalogues of indoles were synthesized from 2-PFPPA.44,49 Thereaction of 2-PFPPA with allylamine followed by cyclizationusing lithium diisopropylamide (LDA) as a base anddeprotection of the indole nitrogen gave 3-Me-indole-f4 in72% from 2-PFPPA (eq 8).

The SeO2 oxidation of N-Ac-3-Me-indole-f4 5 gave indole-f4-3-CHO in 86% yield (eq 9). The SeO2 oxidation of 5 in the

Scheme 4. Synthesis of Trifluoroleucine andTrifluoronorleucine from 2-TFMPA and 3-TFMPA viaAzlactones



presence of acetic anhydride gave 1-Ac-3-(AcO-methyl)indole-f4 6 in 60% yield (eq 9). 3-(AcO-methyl)indole-f4 6 and indole-f4-3-CHO were very useful intermediates for the synthesis oftetrafluoro analogues of tryptophan, tryptamine, and indole-acetic acid.44

Thus, tryptamine-f4 was synthesized from indole-f4-3-CHOin 83% overall yield through condensation with nitromethane,followed by LiAlH4 reduction, while tryptophan-f4 was obtainedin four steps in 51% overall yield through Erlenmeyer’sazlactone method (eq 10). The reaction of 6 with piperidine

gave 3-(piperidinomethyl)indole-f4 in 97% yield, which was aknown key intermediate for tryptophan-f4 in 2 steps (eq 11).Also, indoleacetic acid-f4 was synthesized from 6 in four stepsvia 3-(cyanomethyl)indole-f4 (eq 11).Since it was shown that tryptophan-f4 strongly inhibits both

the tryptophanyl hydroxamate and aminoacyl t-RNA forma-tion,50,51 the tetrafluoro analogues of tryptamine, indoleaceticacid, and other indole derivatives were expected to possessunique physiological activities. Tryptophan-f4 has been used inenzymology, protein engineering and chemical biology.52,53

3. CARBONYLATIONS OFα-(TRIFLUOROMETHYL)VINYL BROMIDE3.1. Synthesis of 2-Trifluoromethylacrylic Acid

through Pd-Catalyzed Carboxylation. The brominationof TFP promoted by photoirradiation followed by dehydro-bromination on KOH gave 2-Br-TFP in high yield.54 Thecarboxylation of 2-Br-TFP catalyzed by a Pd catalyst, e.g.,PdC12(PPh3)2 or PdC12(dppf), in the presence of Et3N inDMF or THF afforded 2-(trifluoromethyl)acylic acid (2-TFMAA) in 65−78% yield (eq 12).55

A variety of trifluoromethacrylates, CH2C(CF3)COOR,were readily prepared from 2-TFMAA, which were potentiallyvery useful monomers for fluorine-containing polymethacry-lates (Figure 1). Copolymerizations with other olefins, e.g.,methyl methacrylate (MMA) and styrenes, were also possible.In fact, the homo- and copolymerizations of methyltrifluoromethacrylate (MTFMA) were reported56 with regardto the development of new radiation-sensitive polymers forresists in microelectronic fabrication processes, whereinMTFMA was prepared from trifluoroacetone. Copolymers ofTFMA esters with styrene and substituted styrenes were alsoprepared.57 We synthesized a variety of new trifluorometha-crylates bearing polyfluoroalkyl ester moieties,58,59 which wouldserve as monomers for potential photoresists and as a

component of optical fibers. Although we did not follow upthis line of research in my laboratory, TFMA esters have beenextensively studied and are still under active investigation forthe development of photoresists for lithography using shortwavelength light, e.g., 157/193 nm,60−62 as we envisioned inthe 1980s.24,58

3.2. α-Trifluoromethyl-β-alanine (α-TFM-β-Ala). Wefound that the addition of gaseous ammonia to 2-TFMAA at0−5 °C in CH2Cl2 gave a novel β-amino acid, α-trifluoromethyl-β-alanine (α-TFM-β-Ala), in excellent yield.44

However, the reaction sometimes gave double and tripleMichael addition products, depending on the reactionconditions. The use of hexamethyldisilazane (HMDS) in anattempt to protect the C-terminus of TFMAA with a TMSgroup resulted in an addition of H2NTMS, generated in situ, toO-TMS-TFMAA, giving N,O-bis-TMS-α-TFM-β-Ala (7) inquantitative yield. No trace of N,N,O-tris-TMS-α-TFM-β-Alawas detected. Also, Michael addition of HMDS to the methyland benzyl esters of TFMAA did not proceed at all. Thedisilylated α-TFM-β-Ala (7), thus obtained, was treated withMeOH to give α-TFM-β-Ala in nearly quantitative yield (eq13).44

α-TFM-β-Ala did not exhibit any antibacterial activity in ourpreliminary screening. However, an enkephalin analoguebearing α-TFM-β-Ala, Tyr-D-Ala-(α-TFM-β-Ala)-Phe-Met, hasshown fairly strong analgesic effects.63,64 This suggested thatthe novel fluoro-β-amino acid would serve as a modifier for avariety of peptide hormones and other physiologically activepeptides, although a method to obtain enantiopure material wasneeded. It should be noted that α-TFM-β-Ala is nowcommercially available from more than several vendors.

3.3. “Ureidocarbonylatlon” of 2-Bromotrifluoropro-pene (2-Br-TFP) Catalyzed by a Pd−Phosphine Com-plex. The Pd-complex-catalyzed amidation of vinyl halides wasshown to be a convenient method for the synthesis of α,β-unsaturated amides.65 However, nothing was known for the Pd-catalyzed reaction of vinyl halides with ureas instead of amines.Accordingly, we investigated the Pd-catalyzed carbonylationreaction of 2-Br-TFP with a urea. Our hypothesis was that bothnitrogen termini of a urea would possess sufficient nucleophil-icity so that the reaction should give the dihydrouracil skeleton

Figure 1. Homo- and block copolymers of 2-TFMAA esters.



in one step. Actually, the reaction proceeded as anticipated togive 5-CF3-5,6-dihydrouracil 8 in good yield, and this novelcarbonylation process was termed “ureidocarbonylation”. Ageneral scheme for this novel process is shown in eq 14.66

However, when unsubstituted urea was employed, the yieldof 8d (R1 = R2 = H) was low. 5-CF3-dihydrouracils 8, thusobtained, were readily converted to the corresponding 5-CF3-uracils 16 by treating with bromine67 in nearly quantitativeyields (eq 9).66 It should be noted that 8 exhibited substantialantitumor activity against ascitic mastocarcinoma MM2 cells.55

A proposed mechanism for the “ureidocarbonylation” isillustrated in Scheme 5.66

We also found a simple method for the synthesis of 8 as wellas its thio analogues just by heating a mixture of 2-TFMAA anda urea or thiourea in the presence of acetic anhydride at 80−100 °C, which afforded the corresponding 8 or its thioanalogues in 50−84% yield (eq 15).55,68 Most importantly, 5-CF3-5,6-dihydrouracil (8d) was obtained in 67% yield(unoptimized) using this method, which was converted to 5-CF3-uracil (9d) in excellent yield.55,68

It is worthy of note that the processes for producing 2-TFMAA from 2-Br-TFP as well as 9d from 2-TFMAA and ureafollowed by dehydrogenation were developed as commercialprocesses by Japan Halon (now Tosoh F-Tech). Furthermore,9d was successfully applied to the commercial synthesis oftrifluridine (trifluorothymidine), an antiherpes antiviral drug,69

primarily used on the eye topically, such as “Viroptic”. This

commercial process was developed by Japan Halon and TokyoYuki Gosei Kogyo in Japan in the early 1990s and is stilloperating at present.

4. HYDROFORMYLATION−AMIDOCARBONYLATIONOF FLUORO-OLEFINS: HIGHLY REGIOSELECTIVEDIRECT SYNTHESIS OF FLUOROAMINO ACIDSFROM FLUORO-OLEFINS4.1. Hydroformylation−Amidocarbonylation of Tri-

fluoropropene (TFP). The hydroformylation−amidocarbony-lation (HF-AC) of TFP was investigated since the reactionshould give the corresponding normal or iso N-acetylaminoacid, Ac-TFNV or Ac-TFV, directly from TFP if the extremelyregioselective hydroformylation was successfully combined withamidocarbonylation. As Scheme 6 shows, the Co-catalyzed

reaction gave Ac-TFNV with 96% selectivity, while the reactioncatalyzed by the Rh−Co binary system (Co2(CO)8/Rh6(CO)16= 50) under the same conditions gave Ac-TFV with 94%selectivity.70 The latter result clearly indicated that the Rh-catalyzed hydroformylation took place exclusively in the firststep to give 2-TFMPA with high selectively, which waseffectively incorporated into the subsequent Co-catalyzedamidocarbonylation.

4.2. Hydroformylation−Amidocarbonylation of Pen-tafluorostyrene (PFS). In contrast to the results obtained forthe reactions of TFP, the attempted regioselective HF-AC ofPFS catalyzed by the Co−Rh binary catalyst system as well asCo2(CO)8 under similar conditions gave unexpected results.The detailed study of the reaction revealed interestingmechanistic aspects of Co−Rh mixed-metal catalyst systems,including a novel CoRh(CO)7-catalyzed process.70

The HF-AC of PFS with acetamide catalyzed by Co2(CO)8gave N-Ac-4-C6F5-homoalanine (10) with 90−92% regioselec-tivity (Scheme 7). This regioselectivity was much higher thanthat (79%) of the simple hydroformylation in benzene.30 Thereaction catalyzed by Co2(CO)8/Rh6(CO)l6 gave N-acetyl-3-C6F5-homoalanine (11) with only ca. 80% regioselectivity,which was much lower than the excellent regioselectivity (98%)

Scheme 5. Mechanism of Pd-Catalyzed Ureidocarbonylationof 2-Br-TFP

Scheme 6. Hydroformylation−Amidocarbonylation of TFP

Scheme 7. Hydroformylation−Amidocarbonylation of PFS



of the simple hydroformylation in benzene.30 To accommodatethese unexpected results, a detailed mechanistic study wasperformed to clarify these anomalies.70

It was found that the hydroformylation was the rate- andregioselectivity-determining step and the presence of acetamidesubstantially increased the normal selectivity, probably byforming an active species HCo(CO)n(CH3CONH2)m. Thiscatalyst species, however, substantially increased the formationof hydrogenation product, C6F5Et. If the Co and Rh catalystsworked independently, the ratio of normal aldehyde formationshould increase at higher Co/Rh ratios and eventually thenormal aldehyde should become the major product. However,contrary to this assumption, an interesting leveling phenomen-on of regioselectivity was observed, i.e., the iso/n ratiodecreases from 94/6 at Co/Rh = 5 to 88/12 at Co/Rh = 25,but the ratio was unchanged (87/13) even at a Co/Rh ratio of100! This leveling phenomenon was best interpreted by takinginto account the formation of and the catalysis by a Co−Rhmixed-metal complex. When we reached this conclusion,Horvath, Bor, and Pino at ETH reported synthesis, character-ization, and some reactions of an interesting coordinativelyunsaturated Co−Rh mixed metal complex, CoRh(CO)7 (eq16),71−73 which was eventually identified as the catalyst speciesin our HF-ADS reaction.

+ ⇌Rh (CO) 2Co (CO) 4CoRh(CO)K

4 12 2 8 71

(16)

In order to directly confirm the catalyst species in dioxane,we performed a high pressure FT-IR study on the Co−Rhmixed metal complex system, which provided strong supportingevidence for the CoRh(CO)7 catalysis.Next, the relative activities of catalyst species were evaluated

on the basis of the equilibrium constant K1 reported for eq14.73 The iso/n ratio vs Co/Rh ratios were calculated andplotted with several given relative catalytic activities. In dioxane,the relative catalytic activity of 54/mol (9/Rh) for Rh6(CO)16/CoRh(CO)7 showed a very good agreement with theexperimental results. The relative catalytic activity forRh6(CO)16/Co2(CO)8 was calculated to be ca. 400000, i.e.,133000 per metal. Finally, kinetic studies were performed tocompare the results with those predicted by calculations basedon the regioselectivity. The kinetic measurements in dioxaneprovided the relative activity values as follows: Rh6(CO)16/CoRh(CO)7 = 3.8/Rh, Rh6(CO)16/Co2(CO)8 = 398000.Consequently, the results of these two independent evaluationmethods were in very good agreement in spite of variousassumptions and simplification for calculations.70

Overall, this study provided a rare and successful example ofthe elucidation of mixed-metal catalysis, in which actual activecatalyst species and their direct precursors were detectedspectroscopically and the observation corresponded almostperfectly to the mechanism proposed based on theregioselectivity analysis. It should be emphasized that thediscovery of CoRh(CO)7 catalysis as well as the successfulmechanistic studies in organometallic chemistry and catalysiswere only possible by the use of unique fluoro-olefin, PFS.From the synthetic viewpoint, a highly regioselective

formation of fluoro-amino acid 12, directly from PFS isnoteworthy. The reaction of PFS using a Co2(CO)8 (5.0 mol%)−Rh4(CO)12 (0.05 mol %) catalyst system gave 12 in 80%yield and 98.2% regioselectivity at 60 °C for 6 h and then 125°C for 5 h under 75 atm of CO and 48 atm of H2.

70 Base-promoted cyclization of 12 gave N-Ac-2-hydroxycarbonyl-3-

methyl-2,3-dihydro-4,5,6,7-tetrafluoroindole (13) in 92% yield,which could be converted to a variety of fluoro-indoles andfluoro-alkaloids (Scheme 8).70

5. APPLICATIONS OFTRIFLUOROMETHYL-CONTAINING AMINO ACIDSTO ENZYME INHIBITORS5.1. Trifluoromethyl Analogues of Captopril as

Inhibitors of Angiotensin-Converting Enzyme. It hasbeen shown that inhibitors of angiotensin converting enzyme(ACE) play key roles in the control of blood pressure astherapeutic agents. Since the development of potent ACEinhibitors captopril74,75 and enalaprilat,76 various analogues ofthese drugs have been designed and synthesized, and their ACEinhibitory activity has been examined. However, little attentionhad been paid to the synthesis and activity of fluorinatedanalogues until we started our investigation in the late 1980s.77

Thus, we designed and synthesized CF3 analogues of captopriland evaluated their ACE inhibitory activity to examine theeffect of fluorine incorporation on the potency.Chiral Michael acceptor 2-CF3-acryloyl-(S)-Pro-OBu-t (14)

was prepared through the facile coupling of (S)-Pro-OBu-t andα-CF3-acryloyl chloride,

55 derived from 2-TMFAA in 85% yield(Scheme 9).78

Conjugate addition of thiolacetic acid to 14 gave adiastereomeric mixture of adducts, (R,S)-15 and (S,S)-15(RS/SS = 1/2) in 70% yield, which were separated by MPLC(Scheme 9).78,79 The stereochemical assignments were

Scheme 8. Tetrafluoroindole Synthesis from PFS via HighlyRegioselective Hydroformylation−Amidocarbonylation

Scheme 9. Synthesis of (R,S)- and (S,S)-Captopril-f 3



unambiguously made on the basis of the X-ray crystal structureof (S,S)-15. Syntheses of (R,S)-captopril-f 3 and (S,S)-captopril-f 3 are illustrated in Scheme 9.78,79

The CF3-analogues of captopril were subjected to in vitroenzyme inhibitory assay against ACE based on the method ofHolmquist et al. using the tripeptide, [3-(2-furyl)acryloyl)]-Phe-Gly-Gly, as the substrate.80 The results are listed in Table1.78,79 (S,S)-Captopril was also used as the reference in thisassay.

As Table 1 shows, (R,S)-captopril-f 3 was found to beextremely potent (IC50 10

−10 M level), while the correspondingdiastereomer, (S,S)-captopril-f 3, was much less potent by 3orders of magnitude. A similar difference in potency wasreported for (S,S)- and (R,S)-captopril, i.e., the (S,S)-isomerwas more potent than the (R,S)-isomer by a factor of 100.74 Itis worthy of note that (R,S)-captopril-f 3 is at least 1 order ofmagnitude more potent than (S,S)-captopril.The considerably higher potency of (R,S)-captopril-f 3 than

(S,S)-captopril may be ascribed to the hydrophobicity and thestereoelectronic effects of the CF3 group. Namely, theincorporation of the (2R)-CF3 group may significantlycontribute to an increase in attractive interaction with thehydrophobic binding site of ACE. Also, the stereospecificincorporation of (2R)-CF3 may cause strong restriction ofrotation around the amide bond because of the stereoelectroniceffect of the CF3 group. This would fix the inhibitor in thefavorable conformation such that strong binding with the activesite is achieved without sacrificing energy for conformationalchange.In fact, the energy minima for hypothetical binding

conformations based on molecular mechanics calculations of(S,S)-captopril and (R,S)-captopril-f 3 indicated the lattercompound to be more favorable by 1.3 kcal/mol, whichcorresponds to 10-fold difference in activity.78,79 Also, the 8.33kcal/mol difference between (R,S)-captopril-f 3 and (S,S)-captopril-f 3 calculated corresponds to the 1,700 times differ-ence in the activity. In addition, a semiempirical approach wasemployed to further examine the inhibitor-enzyme interactionby using the n-SCF-molecular mechanics program (PIMM).81

This calculation indicated that the binding energy of (R,S)-captopril-f 3 was 2.4 kcal/mol more favorable than (S,S)-captopril.87

5.2. Potent Enkephalin Analogues with Trifluoro-methyl-Containing Amino Acid Residues. Enkephalins,“opioid peptides” in the brain, are known to play importantroles as analgesics, regulators of blood pressure, and neuro-transmitters. In the hope of developing nontoxic, nonaddictive,and effective analgesics, replacing morphine, structuralmodifications of enkephalins have been extensively studied,and a variety of analogues have been developed.82 Neuro-biological studies have revealed the presence of the majoropiate receptors μ, δ, and κ that mediate the analgesic effects ofopiates and opioid peptides including enkephalins.83 Thus, thediscovery and development of opiate receptor specific ligands

has been of active interest in medicinal chemistry as well asneurobiology. However, in spite of extensive studies onenkephalin analogues, little attention had been paid to thefluoro-analogues of enkephalins84 when we started ourinvestigation in the early 1990s.The major nemesis of these opioid peptides is a group of

degrading enzymes that cleave the peptide into inactivefragments.85,86 These enzymes include (i) aminopeptidase Mand membrane-bound aminopeptidase(s), cleaving enkephalinsat the Tyr1−Gly2 bond, which is responsible for 80% of thedegradation pathway,85,87 (ii) enkephalinase, cleaving the Gly3-Phe4 bond,86 and (iii) dipeptidylaminopeptidase, cleaving theGly2−Gly3 bond.85 Accordingly, the development of inhibitorsfor these degrading enzymes is an important approach to theenhancement of analgesic activity by increasing the duration ofthe effect.In the course of our study on the synthetic and medicinal

chemistry of CF3-containing amino acids, enzyme inhibitorsand peptide hormones by exploring unique effects of the CF3group,24,35,44,79 we designed and synthesized a series of novelenkephalin analogues bearing TFNV and TFNL. Then, their invivo analgesic activity as well as in vitro receptor binding abilitywas examined.88

Synthesis of Novel Fluoro-enkephalin Analogues. A seriesof fluoro-enkephalin analogues was synthesized through solid-phase peptide synthesis by replacing either Gly2 or Gly3 by (R)-TFNV, (S)-TFNV, or (R)-TFNL, as shown in Scheme 10.88 Ananalogue bearing (N-Me)Phe4 in place of Phe4 was alsosynthesized.

Analgesic Activity Assay in Vivo.88 The in vivo analgesicactivity of fluoro-enkephalin analogues was evaluated by thestandard writhing test using ddY male mice via intracerebro-ventricular (i.c.v.) administration. As Table 2 shows, thesubstitution of Gly2 by (R)-TFNV exhibited a remarkableincrease in potency, i.e., 100000 times stronger thanmethionine−enkephalin, and even 1 order of magnitudestronger than morphine (entry 8). The absolute configurationof TFNV at this position is very important. Thus, [(S)-TFNV2,Met5-NH2]enkephalin showed only ca. 30 times increase inpotency (entry 7). The substitution of Gly3 with TFNVimproved potency by the factor of 30−60, in which the (R)-TFNV analogue was twice as effective as the (S)-TFNVanalogue (entries 10, 11). The modification of carboxylterminus to amide showed 5−6 times improvement in potency

Table 1. ACE Inhibitory Activity of CF3-Analogues ofCaptopril

ACE inhibitor IC50 (M)

(R,S)-captopril-f 3 2.9 × 10−10

(S,S)-captopril-f 3 4.8 × 10−7

(S,S)-captopril 3.6 × 10−9

Scheme 10. Schematic Illustration of Modification Strategyof Methionine−Enkephalin with Trifluoronorvaline (TFNV)and Trifluoronorleucine (TFNL)



(entries 5, 6, 7, and 10). The substitution of Gly2 with (R)-TFNL, a homologue of (R)-TFNV, exhibited 10000 timesincrease in potency, but it was 1 order of magnitude lower thanthe corresponding (R)-TFNV analogue (entry 12). Theobserved remarkable increase in potency for [(R)-TFNV2,Met5-NH2]enkephalin was not entirely exceptional since aknown analogue, [(R)-Ala2, Met5-NH2]enkephalin, showed a10000 fold increase in potency in the same in vivo assay (entry3). In order to assess a “fluorine effect” on potency, (R)-norvaline ((R)-Nval) was synthesized and assayed. As entry 10shows, this analogue exhibited almost equivalent potency tothat of the (R)-Ala2 analogue, which was 1 order of magnitudeweaker than the (R)-TFNV analogue. Accordingly, it is clearthat there was a “fluorine effect”, which improved the potencyby 1 order of magnitude even after a major enhancement factor,i.e., (R)-amino acid residue at Gly2 position, was introduced.The most potent analogue in this series was [(R)-TFNV2, (N-Me)Phe4, Met5-NH2]enkephalin (entry 13), which was 3.5times more potent than [(R)-TFNV2, Met5-NH2]enkephalin.Receptor Binding Assay in Vitro.88 In order to investigate

the origin of the remarkable enhancement in potency by theintroduction of (R)-TFNV at the Gly2 position, the in vitroreceptor binding assays for [(R)-TFNV2, Met5-NH2]enkephalinwere carried out against μ, δ, and κ receptors using tritium-labeled standard ligands. As Table 2 shows, [(R)-TFNV2, Met5-NH2]enkephalin exhibited a 10−10 M level IC50 against μ-receptor, but it was only a half order of magnitudeenhancement in the binding ability compared with methio-nine-enkephalin. For δ-receptor, [(R)-TFNV2, Met5-NH2]-enkephalin showed almost the same level binding ability as

methionine−enkephalin. Interestingly, [(R)-TFNV2, Met5-NH2]-enkephalin bound to the κ-receptor at the 10−7 M levelIC50, whereas methionine−enkephalin did not show anyappreciable binding.The results clearly indicate that the observed remarkable

enhancement in the in vivo potency of [(R)-TFNV2, Met5-NH2]enkephalin was not based on much stronger binding toreceptor sites, but mainly due to the extremely efficientinhibition of degradation by aminopeptidase(s). Possibleenhancement of the rates of absorption and transport, arisingfrom the lipophilicity of CF3 group should also be taken intoaccount as the secondary effect. It is worth noting that [(R)-TFNV2, Met5-NH2]enkephalin was found to cross the blood−brain barrier.The studies described in this section clearly demonstrated

the uniqueness and usefulness of fluoroamino acids as modifiersof peptides and enzyme inhibitors of medicinal interest. Thus,we envisioned that further research in this direction wouldexplore newer and exciting aspects of organofluorine chemistryin medicinal chemistry and chemical biology.89

6. ENANTIOPURE FLUORINATEDα-HYDROXY-β-AMINO ACIDS, THEIR DERIVATIVES,DIPEPTIDES, AND PEPTIDOMIMETICS

β-Amino acids have been attracting considerable interestbecause of their inherent biological activities and their usefulcharacteristics as building blocks for potential therapeutic drugsand “β-peptides” with unique properties.90,91 β-Amino acids arealso useful for the studies of enzymatic reaction mecha-nisms.90,91 Among various types of β-amino acids, α-hydroxy-β-amino acids (isoserines) are one of the most importantmembers because many of them act as potent enzymeinhibitors and they also serve as crucial building blocks forthe compounds of biological and medicinal importance.91,92

For example, α-hydroxy-β-amino acid moieties are found inpaclitaxel93−95 (antitumor agent), bestatin96,97 (inhibitor ofaminopeptidases, immunological response modifier), amasta-tin98 and phebestin99 (aminopeptidase inhibitor), microginin100

(ACE inhibitor, KNI inhibitor), and kinostatins (HIV-1protease inhibitors)101,102 (Figure 2).In the last two decades, substantial research efforts have been

made on the synthesis of fluorinated analogues of β-aminoacids and investigation into their biological implications.103−107

Because of the unique properties of fluorine as an element, theintroduction of fluorine(s), CF2H, or CF3 group to biologicallyactive molecules often critically improves their pharmacologicalproperties.19,103 Moreover, the sensitivity of 19F NMR spec-

Table 2. In Vivo Analgesic Activity of FluoroenkephalinAnalogs (i.c.v.)

entry enkephalin ED50 (10−9 mol/mouse)

1 methionine-enkephalin 7002 morphine·HCl 0.073 Tyr-(R)-Ala-Gly-Phe-Met-NH2 0.054 sedapain (morphine analogue) 0.055 Tyr-(S)-TFNV-Gly-Phe-Met 1206 Tyr-Gly-(S)-TFNV-Phe-Met 1407 Tyr-(S)-TFNV-Gly-Phe-Met-NH2 258 Tyr-(R)-TFNV-Gly-Phe-Met-NH2 0.0079 Tyr-(R)-Nval-Gly-Phe-Met-NH2 0.0410 Tyr-Gly-(S)-TFNV-Phe-Met-NH2 2211 Tyr-Gly-(R)-TFNV-Phe-Met-NH2 1212 Tyr-(R)-TFNL-Gly-Phe-Met-NH2 0.0713 Tyr-(R)-TFNV-Gly-(N-Me)Phe-Met-NH2 0.002

Figure 2. Representative biologically active compounds of medicinal interest bearing an α-hydroxy-β-amino acid residue.



troscopy along with large 19F−1H coupling constants and thevirtual absence of 19F in the living tissue render fluorineincorporation a particularly powerful tool for the investigationof biological processes.8,108,109 Therefore, fluorine-containing α-hydroxy-β-amino acids are expected to serve as useful bioactivecompounds with a wide range of potential applications inmedicinal chemistry and chemical biology. However, only a fewmethods had been reported for the synthesis of fluorine-containing α-hydroxy-β-amino acids when we started ourinvestigation in the mid-late 1990s.19,110−114

Accordingly, newer and efficient approaches to enantiopurefluorine-containing α-hydroxy-β-amino acids needed to bedeveloped. To this end, the “β-lactam synthon method”invented in my laboratory and developed by us andothers115−120 offered an attractive protocol for the synthesisof enantiopure CF2H- and CF3-containing α-hydroxy-β-aminoacids and their congeners.6.1. Synthesis of Enantiopure 3-Hydroxy-4-CF2H-β-

lactams. Racemic cis-β-lactam (±)-16 was prepared through[2 + 2] ketene−imine cycloaddition in good yield (Scheme 11).

Then, the enzymatic optical resolution was carried out usingthe “PS-Amano” lipase.121 This enzyme selectively hydrolyzesthe acetate moiety of (−)-β-lactam (−)-16 to afford kineticallyresolved (3R,4S)-3-AcO-β-lactam (+)-16 (>99% ee) and(3S,4R)-3-hydroxy-β-lactam (−)-17 (96−99% ee) with ex-tremely high enantiopurity in high recovery yields (Scheme11).122,123

Since the acetyl group was not tolerated in thediethylaminosulfur trifluoride (DAST) reaction, the protectinggroup of the 3-hydroxyl moiety of β-lactam (+)-16 wasreplaced with triisopropylsilyl (TIPS). The resulting 3-TIPSO-β-lactam (+)-18 was subjected to ozonolysis to give 4-formyl-β-lactam (+)-19, which was immediately reacted with DAST toafford the corresponding 1-PMP-4-CF2H-β-lactam (+)-20 inhigh yield. Finally, the PMP group was removed using ceriumammonium nitrate (CAN) to give enantiopure (3R,4R)-3-TIPSO-4-CF2H-β-lactam (+)-21 (Scheme 12).122,123 In asimilar manner, (3S,4S)-3-hydroxy-β-lactam (−)-17 wasconverted to enantiopure (3S,4S)-3-TIPSO-4-CF2H-β-lactam(−)-21.6.2. Synthesis of Enantiopure 3-Hydroxy-4-CF3-β-

lactams. A different strategy was employed for the synthesisof enantiopure 4-CF3-β-lactams. Namely, the CF3 moiety wasintroduced, from the very beginning, to the imine to be used forthe [2 + 2] ketene−imine cycloaddition. N-PMP-trifluoroace-taldimine (22) was reacted with the ketene generated in situ

from benzyloxyacetyl chloride to afford racemic cis-3-BnO-4-CF3-β-lactam (±)-23 in moderate yield, as reported by us andothers.111,124 Hydrogenolysis of β-lactam (±)-23, followed byacetylation, gave the corresponding racemic cis-3-AcO-β-lactam(±)-25 in good overall yield (Scheme 13).122,123

The enzymatic optical resolution of β-lactam (±)-25 wasperformed using PS-Amano at 25 °C, pH 7 for 3 h (Scheme14), which gave (3R,4R)-3-AcO-4-CF3-β-lactam (+)-25 with99.9% ee as well as (3S,4S)-3-hydroxy-4-CF3-β-lactam (-)-26with 97% ee in good to high yield.122

3-AcO-4-CF3-β-lactam (+)-25 and 3-hydroxy-4-CF3-β-lactam(−)-26, thus obtained, were converted to the corresponding 3-TIPSO-4-CF3-β-lactams, (+)-28 and (−)-28, using the sameprotocol as that described for the preparation of (+)- and(−)-21 (Scheme 15).122,123

6.3. Synthesis of Enantiopure 1-Acyl-3-hydroxy-4-Rf-β-lactams. The β-lactam synthon method has been developedby exploiting the unique nature of this strained four-memberedskeleton for its facile ring-opening reactions with a variety ofnucleophiles.115,116 When the nitrogen of this strained cyclicamide is acylated (including carbalkoxy, carbamoyl, thiocarba-moyl, and sulfonyl groups besides the standard acyl groups),the resulting N-acyl-β-lactam becomes exceptionally reactive for

Scheme 11. Preparation of Racemic 3-AcO-4-isobutenyl-β-lactam via Staundinger Ketene−Imine Cycloaddition and ItsSubsequent Enzymatic Optical Resolution

Scheme 12. Transformation of 3-AcO-4-isobutenyl-β-lactamto 3-TIPSO-4-CF2H-β-lactam

Scheme 13. Preparation of 4-AcO-4-CF3-β-lactam viaStaudinger Ketene−Imine Cycloaddition

Scheme 14. Efficient Enzymatic Resolution of Racemic 3-AcO-F-CF3-β-lactam



nucleophilic attacks, leading to facile ring-opening coupling.This unique feature of N-acyl-β-lactams has been successfullyutilized in organic synthesis and medicinal chemistry.115,117−120

We prepared a series of N-acyl-3-TIPSO-3-Rf-β-lactams 29(Rf = CF2H or CF3), as examples, in good to high yield throughacylation (including carbalkoxylation and sulfonylation) of 3-TIPSO-4-CF2H-β-lactam 21 and 3-TIPSO-4-CF3-β-lactam 28,using (Boc)2O, ClCO2Bn, 4-F-C6H4COCl and p-TolSO2Cl inthe presence of an appropriate base such as DMAP/Et3N inCH2Cl2 (eq 17: only (3R,4R) series is shown forsimplicity).122,123

6.4. Synthesis of Enantiopure β-CF2H- and β-CF3-α-hydroxy-β-amino Acids and Esters via Facile Hydrolysis/Alcoholysis of N-Acyl-β-lactams. Enantiopure β-Rf-α-hydroxy-β-amino acids (Rf = CF2H or CF3) were readilyobtained through facile ring-opening hydrolysis of (+)- or(−)-29. For example, the reaction of 29 with KOH in aqueousTHF at ambient temperature gave the corresponding O-protected amino acid 30, in high yield (eq 18: only (2S,3S)series is shown for simplicity).122,123 The O-TIPS protectinggroup could be easily removed by HF/pyridine as needed togive 31.

In a similar manner, enantiopure O-TIPS-β-Rf-α-hydroxy-β-amino acid methyl esters, (2S,3S)- or (2R,3R)-32, wereobtained through a facile methanolysis of (+)- or (−)-29 inthe presence of Et3N and a catalytic amount of DMAP atambient temperature in good to quantitative yield (eq 19: only(2R,3R) series is shown for simplicity).122,123 The O-TIPSprotecting group could be easily removed by HF/pyridine asneeded to give 33.

6.5. Synthesis of Rf-Containing Isoserine Dipeptidesthrough Efficient Ring-Opening Coupling of N-Acyl-β-lactams with α- and β-Amino Esters. The ring-openingcoupling of N-acyl-β-lactam, (+)- or (−)-36, with various α-and β-amino acid esters provided a very easy access to a libraryof Rf-containing isoserine dipeptides. Since these couplingreactions do not need any peptide coupling reagents such asDCC, DIC and EDC, the “atom economy”125 is extremely high.The coupling reactions gave the corresponding Rf-containingisoserine dipeptides, 34 and 35, in good to quantitative yields(Scheme 16: Only (S,S,S) or (S,S) series is shown forsimplicity).122,123

The results described above as well as our previousworks126−128 allow us to envision the versatile utility of theN-acyl-3-PO-4-Rf-lactams (P = hydroxyl protecting group) forthe synthesis of Rf-containing isoserine dipeptides, depsipep-tides, peptidomimetics, and key synthetic building blocks for Rf-containing hydroxyethylene, dihyroxylethylene, and hydroxye-thylamine dipeptide isosteres. Possible transformations of N-carbalkoxy-3-PO-4-Rf-lactams are illustrated in Scheme 17.

Scheme 15. Synthesis of (+)- and (−)-3-TIPS-4-CF3-β-lactams

Scheme 16. Ring-Opening Coupling of 1-Acyl-4-Rf-β-lactamswith α- and β-Amino Acid Esters

Scheme 17. Feasible Transformations of 1-Carbalkoxy-4-Rf-β-lactams to Peptides and Peptidomimetics



7. MEDICINAL CHEMISTRY AND CHEMICAL BIOLOGYOF FLUORINE-CONTAINING TAXOIDS7.1. Paclitaxel and Taxoids. Paclitaxel (Taxol) and its

semisynthetic analogue docetaxel (Figure 3) are two of the

most important chemotherapeutic drugs, currently used for thetreatment of advanced ovarian cancer, metastatic breast cancer,melanoma, nonsmall cell lung cancer, and Kaposi’s sarco-ma.129−131 More recently, these drugs have been used for thetreatment of neck, prostate, and cervical cancers.130,131 Themechanism of action of paclitaxel involves its binding to the β-subunit of α,β-tubulin dimer, accelerating the formation ofmicrotubules. The resulting paclitaxel-bound microtubules aremuch more stable and less dynamic than the natural GTP-bound microtubules, with a growth rate higher than thedisassembling rate. The unnatural growth and stabilization ofmicrotubules causes the arrest of the cell division cycle mainlyat the G2/M stage, activating a cell-signaling cascade thatinduces apoptosis.132,133 Although paclitaxel and docetaxelpossess potent antitumor activity, chemotherapy with thesedrugs encounters a number of undesirable side effects as well asdrug resistance.129−131 Therefore, it is important to developnew taxoid anticancer drugs as well as efficacious drug deliverysystems with fewer side effects, superior pharmacologicalproperties, and improved activity against various classes oftumors, especially against drug-resistant cancers.7.2. The Fluorine Probe Approach: Solution-Phase

Structure and Dynamics of Taxoids. The rational design ofnew generation taxoid anticancer agents would be greatlyfacilitated by the development of reasonable models for thebiologically relevant conformations of paclitaxel. In this regard,we recognized that the design and synthesis of fluorine-containing taxoids would have a very useful offshoot ofproviding us with the capability of studying bioactiveconformations of taxoids using a combination of 19F/1HNMR techniques and molecular modeling.134

The early conformational analysis of paclitaxel and docetaxelin solution largely identified two major conformations, withminor variations between studies.135 Structure I, characterizedby a gauche conformation with a H2′−C2′−C3′−H3′ dihedralangle of ca. 60°, was based on the X-ray crystal structure ofdocetaxel136 and was believed to be commonly observed inaprotic solvents (Figure 4, I).135 Structure B, characterized bythe anti conformation with a H2′−C2′−C3′−H3′ dihedralangle of ca. 180°, was observed in theoretical conformationalanalysis137,138 as well as 2D NMR analyses,139 and found in theX-ray structure of the crystal obtained from a dioxane/H2O/xylene solution (Figure 4, II).140 Despite extensive structuralstudies, no systematic study on the dynamics of these two aswell as other possible bioactive conformations of paclitaxel hadbeen reported when we started our study on this problem. Therelevance of the “fluorine probe” approach to study dynamicproperties prompted us to conduct a detailed investigation into

the solution dynamics of fluorine-containing paclitaxel anddocetaxel analogues.The use of 19F NMR for a variable temperature (VT) NMR

study of fluorine-containing taxoids was obviously advantageousover the use of 1H NMR because of the wide dispersion of the19F chemical shifts that allows fast dynamic processes to befrozen out. Accordingly, F2-paclitaxel SB-T-31031 and F-docetaxel SB-T-3001 were selected as probes (Figure 5) forthe study of the solution structures and dynamic behavior ofpaclitaxel and docetaxel, respectively, in protic and aproticsolvent systems.134

Analysis of the low temperature VT-NMR (19F and 1H) and19F−1H heteronuclear NOE spectra of SB-T-31031 and SB-T-3001 in conjunction with molecular modeling revealed thepresence of equilibrium between two conformers in proticsolvent systems. Interpretation of the temperature dependenceof the coupling constants between H2′ and H3′ for SB-T-31031 indicated that one of these conformers (conformer C,Figure 6 and Figure 7) possessed an unusual near-eclipsedarrangement around the H2′−C2′−C3′−H3′ dihedral angle(JH2′‑H3′ = 5.2 Hz, corresponding to the H2′−C2′−C3′−H3′torsion angle of 124° based on the MM2 calculation) and wasfound to be more prevalent at ambient temperatures.134 Theother one corresponded to the anti conformer (conformer B,JH2′‑H3′ = 10.1 Hz, corresponding to the H2′−C2′−C3′−H3′torsion angle of 178° based on the MM2 calculation) and wasquite closely related to the structure II in Figure 4. Theseconformers were different from the one observed in aproticsolvents (conformer A, H2′−C2′−C3′−H3′ torsion angle of54°) that was related to the X-ray crystal structure of docetaxelrepresented by the structure I in Figure 4.136 Figure 6 showsthe Newman projections for these three conformers and Figure7 the structures of the conformers A, B, and C.134

Figure 3. Chemical structures of paclitaxel and docetaxel.

Figure 4. Paclitaxel conformations in aprotic solvent (I) and inaqueous solution (II).

Figure 5. Paclitaxel and docetaxel fluorine probes for NMR analysis.



Restrained molecular dynamics (RMD) studies presentedevidence for the hydrophobic clustering of the 3′-phenyl and 2-benzoate (Ph moiety) for both conformers B and C. Althoughthe conformer C possessed a rather unusual semieclipsedarrangement around the C2′−C3′ bond, the unfavorableinteraction associated with such a conformation was apparentlyoffset by significant solvation stabilization observed in thecomparative RMD study in a simulated aqueous environmentfor the three conformers. The solvation stabilization term forthe conformer C was estimated to be about 10 kcal/mol greaterthan those for the conformers A and B. Accordingly, the“fluorine probe” approach succeeded in finding a newconformer that had never been predicted by the previousNMR and molecular modeling studies.Strong support for the conformer C was found in its close

resemblance to a proposed solution structure of a water-solublepaclitaxel analog, paclitaxel-7-MPA (MPA = N-methylpyridi-nium acetate)141 wherein the H2′−C2′−C3′−H3′ torsionangle of the N-phenylisoserine moiety is 127°, which is only afew degrees different from the value for the conformer C.Thus, the “fluorine probe” approach has proved highly useful

for the conformational analysis of paclitaxel and taxoids inconnection with the determination of possible bioactiveconformations. The previously unrecognized conformer Cmight be the molecular structure first recognized by the β-tubulin binding site on microtubules.Determination of the Binding Conformation of Taxoids in

Microtubules Using Fluorine Probes. The knowledge of thesolution structures and dynamics of paclitaxel and its analoguesis necessary for a good understanding of the recognition and

binding processes between paclitaxel and its binding site on themicrotubules. Such knowledge would also provide crucialinformation for the design of future generation anticanceragents. However, the elucidation of the microtubule-boundconformation of paclitaxel was critical for the rational design ofefficient inhibitors of microtubule disassembly. The lack ofinformation about the three-dimensional tubulin binding site byprotein X-ray crystallography prompted us to apply our fluorineprobe approach to the determination of the F−F distances inthe microtubule-bound F2-taxoids. The results of such a studyshould provide the relevant distance map for the identificationof the bioactive (binding) conformation of paclitaxel.We successfully applied the fluorine probe approach to the

estimation of the F−F distance in the microtubule-bound F2-10-Ac-docetaxel (Figure 5) using the solid-state magic anglespinning (SS MAS) 19F NMR coupled with the radio frequencydriven dipolar recoupling (RFDR) protocol in collaborationwith L. Gilchrist, A. E. McDermott, K. Nakanishi (ColumbiaUniversity), S. B. Horwitz, and M. Orr (A. Einstein College ofMedicine).107

F2-10-Ac-docetaxel was first studied in a polycrystalline formby the RFDR protocol. Based on the standard simulation curvesderived from molecules with known F−F distances (distancemarkers), the F−F distance of two fluorine atoms in F2-10-Ac-docetaxel was estimated to be 5.0 ± 0.5 Å (Figure 8). This

value corresponded quite closely to the estimated F−Fdistances for the conformers B and C (F−F distance was ca.4.5 Å for both conformers) based on our RMD studies for F2-paclitaxel (SB-T-31031). This means that the microcrystallinestructure of F2-10-Ac-docetaxel was consistent with thehydrophobic clustering conformer B or C, but not with theconformer A in which the F−F distance was ca. 9.0 Å.The microtubule-bound complex of F2-10-Ac-docetaxel

revealed the F−F distance to be 6.5 ± 0.5 Å (Figure 8),which was larger than that observed in the polycrystalline formby ca. 1 Å. It is very likely that the microtubule-boundconformation of F2-10-Ac-docetaxel was achieved by a smalldistortion of the solution conformation, i.e., either conformer Bor C.107

The above account demonstrated the power of the fluorineprobe approach that is evident from its ability to supplyextremely valuable and precise information about both boundand dynamic conformations of biologically active molecules.Such information is especially useful in the absence ofknowledge about the three-dimensional crystal structure oftheir binding site. It is worthy of note that our preliminarystudy on the structure of the protein-bound fluorine-labeled

Figure 6. Newman projections of the isoserine moieties of the threeconformers of F2-paclitaxel identified.

Figure 7. Three conformers of F2-paclitaxel identified.

Figure 8. Determination of the F−F distance in F2-10-Ac-paclitaxelusing the RFDR protocol by solid state MAS 19F NMR spectroscopy.



docetaxel, by means of the Solid State MAS 19F NMR using theREDOR protocol, was the very first to tackle such an importantand challenging problem in the structural and chemical biologyof paclitaxel and tubulin/microtubules, which made a solidfoundation for further investigations along this line (see section7.6).7.3. Second-Generation Taxoids. It has been shown that

a primary mechanism of drug resistance is the overexpression ofABC transporters, e.g., P-glycoprotein, an integral membraneglycoprotein that acts as a drug-efflux pump to maintain theintracellular concentration of drugs below therapeutically activelevel.142 In the course of our extensive studies on the design,synthesis and the structure−activity relationship (SAR) oftaxoid anticancer agents, we discovered second-generationtaxoids that possess 1 order of magnitude better activity againstdrug-sensitive cell lines and more than 2 orders of magnitudebetter activity against drug resistant cell lines.143,144 Severalexamples are shown in Table 3 (see Figure 9 for structures). It

was found that meta-substitution of the benzoyl group at theC2 position substantially increased the cytotoxicity of taxoidsagainst drug-resistant cell lines.144,145 This class of the second-generation taxoids is 3 orders of magnitude more potent thanpaclitaxel and docetaxel against drug-resistant cancer cell lines,expressing MDR phenotype (Table 3).144,145

Because of the aforementioned advantages of introducingfluorine into biologically active molecules, we synthesizedfluorine-containing paclitaxel and docetaxel analogues toinvestigate the effects of fluorine−incorporation on thecytotoxicity and the blockage of known metabolic pathways.

7.4. 3′-Difluoromethyltaxoids and 3′-Trifluoromethyl-taxoids. In the course of our extensive SAR studies of thetaxoid anticancer agents, we have synthesized a good number offluorotaxoids by means of the “β-lactam synthon method” toinvestigate the effects of fluorine on cytotoxicity and metabolicstability.106,107,146 Along this line, second-generation fluorotax-oids possessing CF2H and CF3 groups at the 3′-position weresynthesized and their biological activity evaluated.122,147 Thesynthesis of these fluorotaxoids is shown in Scheme 18. The

Ojima−Holton coupling of (3R,4R)-1-Boc-4-CF2H- and 1-Boc-4-CF3-β-lactams 29a, described above (see section 6.3), with2,10-modified baccatins 36 was carried out at −40 °C in THFusing LiHMDS as a base followed by removal of siliconprotecting groups with HF/pyridine to give the correspondingsecond-generation 3′-CF2H- and 3′-CF3-taxoids 37 in moder-ate to high overall yields.For the synthesis of CF3-taxoids 38B (Rf = CF3), we also

investigated a possible kinetic resolution of racemic β-lactam(±)-29a-B (Rf = CF3) during the ring-opening coupling withenantiopure baccatin 36 under the standard conditions, exceptusing 2.5 equivalents of racemic β-lactam.148 As anticipated, wefound a highly efficient kinetic resolution, affording CF3-taxoids37B with diastereomer ratios of 9:1 to >30:1 (based on 19FNMR analysis) in fairly good overall yields after deprotection.In three cases, only the single diastereomer of correctstereochemistry was obtained exclusively. The observed highlevel kinetic resolution of racemic 1-Boc-β-lactam (±)-29a-B bythe lithium salt of the chiral secondary alcohol moiety at the 13-position of baccatin 37B was nicely explained by the stericapproach control using molecular modeling.The cytotoxicity of selected fluorotaxoids against various

cancer cell lines is shown in Table 4. These fluorotaxoidspossess substantially higher potencies than those of paclitaxeland docetaxel against drug-sensitive cancer cell lines and theirpotency against multidrug-resistant cell lines is more impressive(2 orders of magnitude more potent than paclitaxel onaverage).147

7.5. 3′-Difluorovinyltaxoids. As described above, theintroduction of isobutyl, isobutenyl, CF2H, and CF3 groups tothe 3′-position of taxoids, replacing the phenyl group ofpaclitaxel and docetaxel, has led to the development of highlypotent second-generation taxoids, especially against drug-resistant cancer cell lines, expressing MDR phenotype. Our

Table 3. In Vitro Cytotoxicity (IC50, nM)a of SelectedSecond-Generation Taxoids

taxoid MCF7b NCI/ADRc LCC6-WTd LCC6-MDRe

paclitaxel 1.7 300 3.1 346docetaxel 1.0 235 1.0 120SB-T-1213 0.18 4.0SB-T-1103 0.35 5.1SB-T-121303 0.36 0.33 1.0 0.9SB-T-121304 0.9 1.1 0.9 1.2SB-T-11033 0.36 0.43 0.9 0.8

aThe concentration of compound which inhibits 50% (IC50, nM) ofthe growth of a human tumor cell line after 72 h drug exposure.bHuman breast carcinoma. cMultidrug-resistant human ovarian cancercell line. dDrug-resistance factor. eMultidrug-resistant human breastcarcinoma.

Figure 9. Selected second-generation taxoids.

Scheme 18. Synthesis of 3′-CF2H- and 3′-CF3-taxoids



metabolism studies on 3′-isobutyl- and 3′-isobutenyl-taxoidsdisclosed that the metabolism of second-generation taxoids(SB-T-1214 and SB-T-1103) was markedly different from thatof docetaxel and paclitaxel.149 These taxoids were metabolized(via hydroxylation) by CYP 3A4 of the cytochrome P450 familyenzymes primarily at the two allylic methyl groups of the 3′-isobutenyl group and the methyne moiety of the 3′-isobutylgroup (see Figure 10). This finding was in sharp contrast to the

known result that the tert-butyl group of the C3′N-t-Boc moietywas the single predominant metabolic site for docetaxel.150

These unique metabolic profiles prompted us to design andsynthesize 3′-difluorovinyl-taxoids in order to block the allylicoxidation by CYP 3A4 mentioned above, which should enhancethe metabolic stability and activity in vivo.For the synthesis of a series of 3′-difluorovinyltaxoids 48,

novel (3R,4S)-1-t-Boc-3-TIPSO-4-difluorovinyl-β-lactam(+)-47 was the key component for the coupling with baccatins43 (Scheme 19).151 We prepared β-lactam (+)-47 in threesteps from 4-formyl-β-lactam (+)-26 (see Scheme 1) using theWittig reaction of the formyl moiety with difluoromethylphos-phorus ylide generated in situ from (Me2N)3P/CF2Br2/Zn(Scheme 19).151 The ring-opening coupling reaction of β-lactam (+)-47 with baccatins 43 (X = H, MeO, N3) and thesubsequent removal of the silyl protecting groups gave the

corresponding 3′-difluorovinyltaxoids 41 in good to excellentyields (Scheme 19).151 Cytotoxicities of the 3′-difluorovinyltax-oids 41 were evaluated against 4 human cancer cell lines.151,152

Results for selected taxoids are summarized in Table 5.As Table 5 shows, all difluorovinyltaxoids 41 are exceedingly

potent as compared to paclitaxel. A clear effect of C2-benzoatemodification at the meta position (X = H vs X = MeO or N3)was observed on the increase in potency against MCF7 (Pgp-)and NCI/ADR (Pgp+) cell lines (entries 2−5 vs entries 6−11).Difluorovinyltaxoids with 2,10-modifications (entries 6−11)exhibited impressive potency, exhibiting IC50 values in the <100pM range (78−92 pM) except one case against MCF7 (entry7) and in the subnanomolar range (0.34−0.57 nM) againstNCI/ADR, which was 3 orders of magnitude more potent thanpaclitaxel. The resistance factor for these taxoids is 1.7−6.4,while that for paclitaxel is 250. Difluorovinyltaxoids with anunmodified C2-benzoate moiety (entries 2−5) also showedhighly enhanced potency against MCF7 and NCI/ADR ascompared to paclitaxel. These taxoids exhibited impressivepotency against HT-29 (human colon) and PANC-1 (humanpancreatic) cancer cell lines as well. SB-T-12853 appearedparticularly promising against these gastrointestinal (GI) cancercell lines.

7.6. Possible Bioactive Conformations of Fluorotax-oids. As described in section 7.2, we have successfully usedfluorine-containing taxoids as probes for NMR analysis of the

Table 4. In Vitro Cytotoxicity (IC50, nM)a of Selected 3′-CF2H- and 3′-CF3-taxoids 37taxoid Rf R X MCF7b (breast) NCI/ADRc (ovarian) R/Sd LCC6- WTe (breast) H460f (lung) HT-29g (colon)

paclitaxel 1.7 300 176 3.1 4.9 3.6docetaxel 1.0 215 215 1.0SB-T-12841-1 CF2H Ac N3 0.32 1.68 5.3 0.22 0.48 0.57SB-T-12842-2 CF2H Et-CO F 0.53 7.24 14 0.88 0.41 0.86SB-T-12843-1 CF2H Me2N-CO MeO 0.45 4.51 10 0.69 0.40 0.43SB-T-12844-3 CF2H MeO-CO Cl 0.26 2.08 8.0 0.13 0.25 0.29SB-T-12821-2 CF3 Ac F 0.45 5.58 13 0.38 0.49 1.11SB-T-12822-4 CF3 Et-CO N3 0.38 1.61 4.2 1.09 0.20 0.40SB-T-12823-3 CF3 Me2NCO Cl 0.12 1.02 8.5 0.27 0.42 0.45SB-T-12824-1 CF3 MeOCO MeO 0.17 2.88 17 0.27 0.38 0.53

aConcentration of compound that inhibits 50% (IC50, nM) of the growth of human tumor cell line after a 72 h drug exposure. bMCF7: humanbreast cancer cell line. cNCI/ADR: adriamycin-resistant human ovarian cancer cell line (Pgp+) (originally designated as “MCF7-R). dResistancefactor = (IC50 for drug resistant cell line, R)/(IC50 for drug-sensitive cell line, S). eLCC6-WT: human breast cancer cell line (Pgp-). fHumannonsmall cell lung cancer cell line. gHuman colon cancer cell line.

Figure 10. Primary sites of hydroxylation on the second-generationtaxoids by cytochrome P450 family enzyme 3A4.

Scheme 19. Synthesis of C3′-Difluorovinyltaxoids



conformational dynamics of paclitaxel in conjunction withmolecular modeling.134 We have further applied the fluorine-probe protocol to the SS MAS 19F NMR analysis with theRFDR method to measure the F−F distance in the micro-tubule-bound F2-10-Ac-docetaxel.

107 Then, Schaefer and co-workers used the rotational echo double resonance (REDOR)to investigate the structure of the microtubule-bound paclitaxelby determining the 19F−13C distances of a fluorine-probe ofpaclitaxel (Figure 11).153 These SS MAS 19F NMR studies haveprovided critical information on the bioactive conformation ofpaclitaxel and docetaxel.

In 2005, we proposed a new bioactive conformation ofpaclitaxel, “REDOR-Taxol”,154 based on (i) the 19F−13Cdistances obtained by the REDOR experiment,154 (ii) thephotoaffinity labeling of microtubules,155 (iii) the crystalstructure (pdb code: 1TUB) of Zn2+-stabilized α,β-tubulindimer model determined by cryo-electron microscopy (cryo-EM),156 and (iv) molecular modeling (Monte Carlo; Macro-model).154 In this computational biology analysis, we docked apaclitaxel-photoaffinity label molecule to the position identifiedby our photoaffinity labeling study first and then optimized theposition with a free paclitaxel molecule in the binding spaceusing the REDOR distances as filters.154

In 2007, three additional intramolecular distances of the keyatoms in the microtubule-bound 19F/2H-labeled paclitaxel weredetermined by the REDOR method (Figure 11).157 Also, it hasbeen shown that the optimized cryo-EM crystal structure oftubulin-bound paclitaxel (pdb code: 1JFF)158 serves better forthe computational structure analysis. Accordingly, we haveoptimized our REDOR-Taxol structure, using the 1JFFcoordinates as the starting point, by means of moleculardynamics simulations (Macromodel, MMFF94) and energyminimization (InsightII 2000, CVFF).159

We applied the same computational protocol to investigatethe microtubule-bound structures of the 3′-CF2H-, 3′-CF3-, and3′-CF2CCH-taxoids, using the updated REDOR-Taxol159 asthe starting structure. Three fluoro-taxoids, SB-T-1284, SB-T-1282, and SB-T-12853 (Figure 12), were docked into the

binding pocket of paclitaxel in the β-tubulin subunit bysuperimposing the baccatin moiety with that of the REDOR-Taxol and their energies minimized (InsightII 2000, CVFF).The resulting computer-generated binding structures of threefluoro-taxoids are shown in Figure 13 (A, B, and C).151

As shown in Figure 13 (A, B, and C), the baccatin moietyoccupies virtually the same space in all cases, as expected. Eachfluorotaxoid fits comfortably in the binding pocket without anyhigh-energy contacts with the protein. There is a very stronghydrogen bond between the C2′−OH of a fluorotaxoid andHis229 of β-tubulin in all cases, which shares the same keyfeature with the REDOR-Taxol structure.154 It should be notedthat our preliminary study on the tubulin-bound structures ofthese three fluorotaxoids using the 1TUB coordinates led todifferent structures in which the C2′−OH had a hydrogenbonding to Arg359 of β-tubulin.160 However, the use of theupdated REDOR-Taxol structure based on the 1JFFcoordinates unambiguously led to the fluoro-taxoids structuresbearing a strong hydrogen bond between the C2′−OH andHis229.151,152

The CF2H and CF3 moieties fill essentially the same space, asanticipated. However, the CF2CCH moiety occupies moreextended hydrophobic space than the CF2H and CF3 moieties.It is likely that this additional hydrophobic interaction issubstantially contributing to the exceptional cytotoxicity of 3′-

Table 5. In Vitro Cytotoxicity (IC50, nM)a of 3′-Difluorovinyltaxoids 41entry taxoid R X MCF7b (breast) NCI/ADRc (ovarian) R/S HT-29d (colon) PANC-1e (pancreatic)

1 paclitaxel 1.2 300 250 3.6 25.72 SB-T-12851 Ac H 0.099 0.95 9.6 0.41 1.193 SB-T-12852 c-Pr-CO H 0.12 6.0 50 0.85 5.854 SB-T-12853 Et-CO H 0.12 1.2 10 0.34 0.655 SB-T-12854 Me2N-CO H 0.13 4.3 33 0.46 1.586 SB-T-12852-1 c-Pr-CO MeO 0.092 0.48 5.27 SB-T-12853-1 Et-CO MeO 0.34 0.57 1.78 SB-T-12855-1 MeO-CO MeO 0.078 0.50 6.49 SB-T-12851-3 Ac N3 0.092 0.34 3.710 SB-T-12852-3 c-Pr-CO N3 0.092 0.45 4.911 SB-T-12855-3 MeO-CO N3 0.078 0.40 5.3

a−dSee footnotes of Table 3. eHuman pancreatic carcinoma.

Figure 11. Solid-state NMR studies on microtubule-bound fluorotax-oid probes.

Figure 12. Three fluorotaxoids used for molecular modeling analysis.



difluorovinyltaxoids. The overlay of SB-T-12853 with arepresentative second-generation taxoid, SB-T-1213 showsvery good fit, which may suggest that the difluorovinyl groupmimics the isobutenyl group (Figure 13, D). However, thedifluorovinyl group is in between the vinyl and isobutenylgroups in size, and two fluorine atoms may mimic two hydroxylgroups rather than two methyl groups electronically. Accord-ingly, the difluorovinyl group is a very unique structuralcomponent in medicinal chemistry and may serve as a versatilemodifier of pharmacological properties in a manner similar tothe trifluoromethyl group.7.7. Use of Fluorine in Taxoid-Based Tumor-Targeting

Anticancer Agents. Traditional chemotherapy depends onthe premise that rapidly proliferating tumor cells are more likelyto be destroyed by cytotoxic agents than normal cells. In reality,however, these cytotoxic agents have little or no specificity,which leads to systemic toxicity causing undesirable side effects.Accordingly, the development of tumor-specific drug deliverysystems for anticancer agents, differentiating the normal tissuesfrom cancer cells or tissues, is an urgent need to improve theefficacy of cancer chemotherapy. Various drug delivery systemshave been studied over the past few decades to address thisproblem.161 Rapidly growing cancer cells overexpress tumor-specific receptors to enhance the uptake of nutrients andvitamins. These receptors can be used as targets for cancercell−specific delivery of cytotoxic agents through receptor-mediated endocytosis. Furthermore, the characteristic physiol-ogy of tumor and cancer cells can be exploited to selectivelyaccumulate and release a cytotoxic agent inside these cells.Monoclonal antibodies, polyunsaturated fatty acids, folic acid,biotin, aptamers, transferrin, oligopeptides, and hyaluronic acid,for example, have been employed as tumor-specific “guidingmodules” to construct tumor-targeting drug conjugates.161−166

As a general structure, tumor-targeted drug delivery systemsconsist of a tumor-targeting module (TTM) conjugated to acytotoxic warhead directly or through a suitable “smart” linker(Figure 14). These drug conjugates should be stable in bloodcirculation to minimize systemic toxicity and should beeffectively internalized inside the target tumor cells. Uponinternalization, the drug conjugate should efficiently release thecytotoxic agent without loss of potency. Thus, the “smart”

linkers should possess proper characteristics to provide suitablestability and reactivity.Fluorine-containing anticancer agents can certainly serve as

potent “warheads” for the tumor-targeting drug conjugates.104

In addition, fluorine can be strategically incorporated to createuseful biochemical tools that facilitate the development of suchtumor-targeting drug conjugates. For example, 19F NMR canprovide techniques to directly observe time-dependentprocesses in complex biological systems since the fluorinenucleus is virtually nonexistent in biological systems.167 As 18Fis a common radioisotope used for positron emissiontomography (PET) imaging, selective fluorination for biodis-tribution studies has found extensive use in diagnosticapplications.168−170

This section concisely describes our recent approaches to thestrategic incorporation of fluorine(s) into taxoid-based tumor-targeted drug delivery systems and ongoing investigations withfuture perspective.

7.7.1. Polyunsaturated Fatty Acid (PUFA)−Taxoid Con-jugates. Omega-3 polyunsaturated fatty acids (PUFA), such aslinolenic acid (LNA), eicosapentaenoic acid (EPA), anddocosahexaenoic acid (DHA), are naturally occurring com-pounds found in vegetable oils, cold-water fish, and meat.171

Perfusion studies have shown that some PUFAs are taken upmore rapidly by tumor cells than normal cells.172 It has alsobeen shown that PUFAs are readily incorporated into cellularmembranes, catabolized as an energy resource, and producemetabolites that act as signaling molecules, modulating variousintracellular processes ranging from inflammatory response tocellular proliferation.173 It has been shown that the conjugationof a drug to a PUFA greatly alters the pharmacokinetics (PK)profile of the parent compound, leading to tumor-selectiveaccumulation of the drug conjugate.174 Thus, a number ofPUFA−drug conjugates are currently undergoing preclinicaland clinical evaluations.175 For example, DHA−paclitaxel(Taxoprexin), currently in phase III clinical trials, has exhibitedbetter efficacy than paclitaxel in some studies174,176 but doesnot show efficacy against multidrug-resistant (MDR) tumorsthat overexpress P-glycoprotein. As metioned above, second-generation taxoids and fluorotaxoids exhibited 2−3 orders ofmagnitude higher potency than paclitaxel against MDR cancercell lines. Thus, PUFA conjugates, bearing a second-generationtaxoid or fluorotaxoid should be more efficacious than DHA-paclitaxel against drug-resistant tumors.Accordingly, novel DHA− and LNA−taxoid conjugates were

synthesized and assayed in vivo for their efficacy against

Figure 13. Computer-generated protein-bound structures of 3′-Rf-taxoids in β-tubulin 1JFF: (a) SB-T-1284 (3′-CF2H); (b) SB-T-1282(3′-CF3); (c) SB-T-12853 (3′-CF2CH); (d) overlay of SB-T-12853and SB-T-1213 (3′-isobutenyl).

Figure 14. General structures of tumor-targeted drug delivery systems.



different drug-resistant and drug-sensitive human tumorxenografts in severe combined immune deficiency (SCID)mice. Several of these conjugates led to a complete regressionof the tumor in all surviving mice with minimal systemictoxicity. For example, DHA-SB-T-1214 led to a completeregression of the highly drug-resistant DLD-1 colon tumorxenograft in mice without appreciable systemic toxicity, whereinno recurrence of tumor growth was observed for more than 190days after treatment.177 DHA-SB-T-1214 is currently under-going extensive late-stage preclinical evaluation, and anInvestigational New Drug (IND) application will be filed tothe US FDA in the near future. Since 3′-difluorovinyltaxoidsexhibit equivalent or even higher potency than SB-T-1214 withexcellent metabolic stability, DHA and LNA conjugates of SB-T-12854 (Figure 15) have been synthesized and their efficacy

in vivo is currently under active investigation. A preliminary invivo efficacy study on LNA-SB-T-12854 against highlymetastatic MX-1 human breast tumor xenograft in nude micehas shown promising results, wherein the complete eradicationof tumor was achieved using weekly regimen for drugadministration.7.7.2. Self-Immolative Disulfide Linkers for Tumor-

Targeted Drug Delivery. Monoclonal Antibody−TaxoidConjugates with First-Generation Disulfide Linker. Cancercells overexpress certain antigens on the cell surface and thesetumor-specific antigens can be used as biomarkers todifferentiate tumor tissues from normal tissues.161,178,179

Certain monoclonal antibodies (mAb) have high bindingspecificity to tumor-specific antigens and can be used as drugdelivery vehicles to carry a payload of cytotoxic agentsspecifically to the tumor site. The mAb−drug conjugate isinternalized upon binding to the tumor antigen via receptor-mediated endocytosis (RME) and the payload is released insidethe cancer cell. For example, brentuximab vedotin (Adcetris) isan mAb−drug conjugate targeting CD30 and recently approvedby the FDA for the treatment of Hodgkin’s lymphoma, andseveral other mAb−drug conjugates are currently in humanclinical trials.180,181

The efficacy of mAb−drug immunoconjugates depends notonly on the specificity of the mAb and the potency of thecytotoxic drug, but also on the linker which connects the mAbto the drug. We successfully conjugated a highly cytotoxic C-10methyldisulfanylpropanoyl taxoid to immunoglobin G classmAbs, recognizing the epidermal growth factor receptor(EGFR), through a disulfide-containing linker (Figure 16).182

These conjugates showed excellent selectivity in vitro andremarkable antitumor activity in vivo against A431 humansquamous tumor xenografts in SCID mice, resulting in

eradication of the tumor without appreciable systemictoxicity.182 However, the modification at the 10 position ofthe taxoid resulted in 8−10 times loss of potency relative to theparent taxoid.182 Accordingly, the mechanism-based second-generation linker system was designed and developed to allowthe release of the unmodified taxoid with uncompromisedpotency.

Second-Generation Self-Immolative Disulfide Linkers.Second-generation mechanism-based bifunctional disulfidelinkers can be generally used to connect a warhead to oneend and a tumor-targeting module (TTM) to the other end.This self-immolative disulfide linker module can release a taxoidwarhead efficiently inside cancer cells by taking advantage of1000 times higher concentration of glutathione in tumor ascompared to that in blood plasma.183 When the TTM navigatesthe drug-conjugate to the target receptors on the tumor surface,the whole conjugate is internalized via RME. Then, anintracellular thiol-triggered cascade drug-release takes placethrough thiolactonization (Figure 17) and the released potent

anticancer drug attacks its target protein, i.e., microtubules fortaxoids. To promote the thiolactonization process, a phenylmoiety was strategically placed to direct the cleavage of thedisulfide bond by intracellular thiol (e.g., glutathione),generating a thiophenolate or sulfhydrylphenyl species whichattacks the ester linkage to the drug molecule (Figure 18).The validity of this self-immolative drug-release mechanism

has been proven in a model system using fluorine-labeling andmonitoring by 19F NMR spectroscopy (Figure 18)104 as well asin a real system with cancer cells using fluorescence-labelingand confocal fluorescence microscopy (CFM).184 These self-immolative disulfide linkers have been successfully incorporatedinto various tumor-targeting drug conjugates and their efficacyevaluated in cancer cells.184−186

Figure 15. Omega-3 polyunsaturated fatty acid−fluorotaxoid con-jugates.

Figure 16. mAb−taxoid conjugate with first-generation disulfidelinker.

Figure 17. Second-generation self-immolative disulfide linker.



7.7.3. Vitamins as Tumor-Targeting Modules. Cancer cellscritically require certain vitamins, such as those essential for celldivision, to sustain their rapid growth although vitamins areessential for the growth and development of all living cells.Receptors for vitamins, including those for folic acid (vitaminM, vitamin B9), biotin (vitamin H, vitamin B7, coenzyme R),and vitamin B12, are overexpressed on the cancer cell surface,providing useful targets for tumor-targeted drug delivery.187

Among those vitamin receptors, folate receptors have beenwell-established and extensively studied as the target for drugdelivery.188 Biotin serves as a coenzyme for five biotin-dependent carboxylases and plays a significant role in epigeneticregulation, fatty acid synthesis, energy production and themetabolism of fats and amino acids.189,190 Biotin receptors hadnot been studied until 2004 when vitamin receptor-targetedrhodamine polymers indicated that receptors for biotin wereeven more overexpressed on the surface of cancer cells thanthose for folic acid and vitamin B12.

187 Thus, the biotin receptorhas emerged as a novel target for tumor-targeted drug deliveryin addition to the well-established folate receptors.A general mechanism is illustrated in Figure 19 for the

internalization of a tumor-targeting drug conjugate throughreceptor-mediated endocytosis (RME), drug release, and thebinding of the released drug to the target protein (taxoid binds

microtubules).185 The internalization of the fluorescent probesvia RME, drug release via disulfide bond cleavage and bindingof the released taxoid “warhead” to microtubules were validatedby CFM analysis of three fluorescent probes, i.e., (a) biotin−FITC conjugate, (b) biotin−linker−coumarin conjugate and(c) biotin−linker−taxoid(fluorescein).166,184,186In addition, biotin−linker−taxoid conjugate was synthesized

and assayed in vitro against L1210 (mouse lymphocyticleukemia), L1210FR (folate and biotin receptors overexpressedL1210 leukemia) and WI38 (normal human lung fibroblasto-ma) cells to examine the efficacy of the biomarker-specifictargeting of the conjugate.166,184,186 The IC50 values of theconjugate (taxoid = SB-T-1214) against L1210FR, L1210, andWI38 cell lines were 8.80 nM, 522 nM and 570 nM,respectively. The results clearly indicate the high target-specificity of the drug conjugate, which is consistent with theRME-based internalization and drug release observed by CFMand flow cytometry using fluorescent probes. Accordingly, thistumor-targeted drug delivery system bearing a vitamin as thetumor-targeting module is ready to utilize highly potentfluorotaxoids as “warheads”.

Vitamin−linker−taxoid Conjugates Bearing an ImagingArm for PET Analysis. As a part of our approach to developingtaxoid-based novel “theranostics”, i.e., combination of diag-nostics and therapy, we designed vitamin−linker−taxoidconjugates bearing an imaging arm for PET analysis (Figure20). In this novel drug conjugate, a vitamin TTM and taxoid

“warhead” are linked via a 1,3,5-triazine splitter with a water-solubilizing triethylene glycol (PEG3) spacer and a self-immolative disulfide linker. The imaging arm is attached tothe triazine splitter using click chemistry. The 18F-(PEG3)-moiety can be introduced either through the click reaction of18F-(PEG3)-N3 with the propargylaminotriazine conjugate or18F fluorination of the MsO-(PEG3)-triazole linked to thetriazine splitter in the drug conjugate, with BuN18F. For afeasibility study, we have successfully synthesized a prototypeconjugate with a 19F-(PEG3)-arm, using biotin as TTM and SB-T-1214 as “warhead”. The corresponding “hot” chemistryexperiment will be performed in collaboration with the Fowlerlaboratory at the Brookhaven National Laboratory, shortly.Results of this radio-synthesis and biodistribution analysis ofthe tumor-targeting drug conjugate in real-time will be reportedelsewhere in due course. Again, fluorotaxoids would serve aspowerful “warheads” for these “theranostics” as well.

7.7.4. Nanoscale Vehicles for Tumor-Targeted DrugDelivery. Single-Walled Carbon Nanotubes as Vehicles for“Trojan Horse” Tumor-Targeted Drug Delivery. In the pastdecade, carbon nanotubes (CNTs) have emerged as a uniqueand efficient vehicle for transport and delivery of drugs.191,192

Since functionalized carbon nanotubes ( f-CNTs) were found

Figure 18. Time-dependent monitoring of disulfide cleavage andthiolactonization by 19F NMR in a model system (adapted from ref104. Copyright 2004 Wiley-VCH).

Figure 19. Schematic representation of the RME of a drug conjugate,drug release, and drug binding to the target protein (adapted from ref186. Copyright 2010 American Chemical Society).

Figure 20. Vitamin−linker−taxoid conjugates bearing an imaging armfor PET analysis.



to be nonimmunogenic and exhibit low toxicity, drug deliverysystems using f-CNTs have been extensively studied.191,192 Wethought that single-walled carbon nanotubes (SWNTs)functionalized with vitamins as TTM would provide ananoscale and biocompatible platform for tumor-targeteddrug delivery. Thus, we designed and synthesized a novelbiotin−SWNT−linker−taxoid(fluorescein) conjugate 42 (Fig-ure 22) to investigate the mass-delivery of payloads to cancercells, wherein the enhancement of internalization via RME wasalso expected through multivalent binding of TTM to thevitamin receptors.184

The internalization via RME, drug release and binding to thetarget protein (i.e., microtubules) of fluorescent SWNT−taxoidconjugate 42 were confirmed using CFM (Figure 21) andquantified by flow cytometry analysis using L1210FR cells.184

The results were consistent with those for the biotin−linker−taxoid(fluorescein) conjugate mentioned above.The cytotoxicity assay of the conjugate 42 against L1210FR,

L1210 and WI38 cell lines (IC50 0.36, >50, and >50 μg/mL,respectively) has revealed excellent target-specificity andsubstantially enhanced potency attributed to mass-delivery ofthe taxoid warheads inside the cancer cells. The results ensurethe merit of the “Trojan Horse” strategy in tumor-targetingdrug delivery.184 This nanoscale drug delivery system isattractive for the tumor-specific delivery of highly potentfluorotaxoids. In addition to the use of a fluorotaxoid as“warhead”, we plan to investigate possible monitoring of thedrug release by 19F NMR, using SWNT-taxoid conjugate 43bearing a fluorine-containing self-immolative disulfide linker,wherein an unmodified “warhead” is used instead offluorescein-tethered taxoid (Figure 22).Asymmetric Bowtie Dendrimers As Vehicles for Tumor-

Targeted Drug Delivery. Dendrimers are monodisperse,starburst polymers with a defined architecture originatingfrom a central core. The high density of surface functionalgroups allows for polyvalent conjugation of TTMs, drugs, andtracers, which has made dendrimers highly versatile vehicles fortumor-targeted drug delivery, and extensive studies have beendone.193 Recently, an orthogonal diblock strategy for thegeneration of asymmetric bowtie dendrimers with a polyamido-(amine) (PAMAM) backbone and a cystamine core has beendeveloped in our laboratory as well as by others.194 First, twodendrimers are fully functionalized with TTMs, drugs or

imaging agents. Second, each dendrimer is reductively cleavedto produce two half dendrons that can be asymmetrically joinedvia a bis-maleimido spacer. This synthetic approach offersunique advantages of selective modification and quantificationof TTMs, drugs, and tracers on each half-dendron prior tocoupling. Therefore, this strategy should produce tumor-targeting nanoscale drug conjugates with well-defined drugloading and enhanced tumor specificity.We have designed a novel asymmetric bowtie dendrimer

scaffold bearing a 1,3,5-triazine splitter in the bis-maleimidelinker module to introduce an imaging module (Figure 23). Weset out to construct a prototype dendrimer conjugate whereinthe vitamin TTM is biotin, the “warhead” is fluorotaxoid SB-T-12854 or SB-T-1214, and the imaging module is fluorine (19Ffor cold material and 18F for PET). In this prototype, anasymmetric bow-tie dendrimer, consisting of the G3 and G1PAMAM dendrons, is used. The G3 dendron allows theconjugation of 16 biotin molecules, while the G1 dendronholds 4 taxoids. We have successfully synthesized keycomponents and the final assembly to the designed conjugateis in progress. Synthesis and biological evaluation of thisdendrimer-based tumor-targeted drug delivery system will bereported in due course.

■ CONCLUDING REMARKSThis Perspective has covered the evolution of my researchendeavor on fluorine chemistry not as a specialist in this field,but as an explorer of its interfaces with multidisciplinary fields

Figure 21. Novel SWNT-based tumor-targeting “Trojan Horse” drug conjugate 42 and the CFM images of L1210FR cells treated with 42 incubated(A) before and (B) after the addition of GSH-ethyl ester. Image B clearly highlights the presence of fluorescent microtubule networks in the livingcells generated by the binding of taxoid−fluorescein upon cleavage of the disulfide bond in the linker by either GSH or GSH-ethyl ester. (CFMimages adapted from ref 184. Copyright 2008 American Chemical Society).

Figure 22. Novel SWNT-based tumor-targeting “Trojan Horse” drugconjugate 43 bearing a fluorine probe.



in chemistry and biology. As mentioned, I was a syntheticchemist specializing in organometallic chemistry and homoge-neous catalysis when I was brought into a very unique world of“fluorine chemistry” at the end of the 1970s. My laboratorystarted exploring the interface of fluorine chemistry andtransition-metal catalysis, especially hydrocarbonylations andamidocarbonylation, which opened highly efficient syntheticroutes to a variety of organofluorine compounds. Among them,CF3-containing enantiopure amino acids were successfullyapplied to the enzyme inhibitor design, leading to the discoveryof highly potent ACE inhibitor and enkephalin analogues. Thisline of research brought us into the field of medicinal chemistry.Also, trifluoromethacrylic acid was the key compound for thesynthesis of trifluorothymine and this process was incorporatedinto the commercial process for the production of antiviraldrug, trifluridine. We also introduced fluorine chemistry to the“β-lactam synthon method” and demonstrated the versatile androbust utility of fluorine-containing N-acyl-β-lactams as keyintermediates to a library of fluorine-containing α-hydroxyl-β-amino acids and their peptides. Through expansion of thischemistry, we synthesized novel fluorine-containing taxoids andused them as excellent probes for the identification of bioactiveconformations of paclitaxel and taxoids by means of 19F NMRin solution and solid phase. We also designed and developed aseries of fluorine-containing taxoids, which are highly potent invitro and in vivo, especially against multidrug resistant tumorsthrough strategic incorporation of fluorine for potency andmetabolic stability. In the past decade, my laboratory has beendeveloping new and efficacious tumor-targeted drug deliverysystems for new generation cancer chemotherapy. These noveldrug delivery systems consist of tumor-targeting modules,mechanism-based self-immolative disulfide linkers and war-heads. Fluorotaxoids are obviously highly potent “warheads” forthese tumor-targeting drug conjugates. In order to monitor thetumor-specific drug delivery, internalization, drug release, anddrug binding to the target protein, we have been exploring the

potential of the “fluorine probe” approach based on 19F NMRas well as 18F-tracer for PET analysis in addition tofluorescence-based probes. The use of multifunctionalizedSWNTs as a drug delivery system bearing multiple-warheadsand multiple-targeting modules has shown highly promisingresults on the benefit of mass-delivery (“Trojan Horse”strategy) of anticancer drug molecules to cancer cells withhigh specificity. We have also been constructing anothernanosacle drug delivery system based on asymmetric bowtiePAMAM dendrimers, bearing an imaging module, including 18Ftracer for PET. Our future efforts will be concentrated on thedesign and synthesis of “tailor made nano-medicines” bearingimaging modules (for MRI, PET, fluorescence imaging and 19FNMR), which would enable us to perform diagnostics andtherapy in the real time.Thus, my laboratory has been exploring the interfaces of

fluorine chemistry and multidisciplinary field of researchinvolving medicinal chemistry, chemical biology, cancerbiology, and molecular imaging. When we explore new areasof research, naturally we enjoy and struggle with many “first”findings, successes, and problems. This is, to my opinion, themost exciting thing as a researcher.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] authors declare no competing financial interest.Biography

Iwao Ojima received his Ph.D. in organic chemistry from theUniversity of Tokyo. He is currently a University DistinguishedProfessor and the Director of the Institute of Chemical Biology &Drug Discovery at Stony Brook University. His broad researchprogram can be summarized as synthetic organic chemistry at thebiomedical interface, including medicinal chemistry, organofluorinechemistry, and chemical biology.

■ ACKNOWLEDGMENTSThe author acknowledges numerous research grants from theNational Institutes of Health (National Institute of GeneralMedical Sciences and National Cancer Institute), NationalScience Foundation, and ACS Petroleum Research Fund, whichhave been supporting his research for the last three decades.Generous support from the New York State Science andTechnology Foundation, Author C. Cope Fund, New YorkState Office of Science, Technology and Academic Research(NYSTAR), Rhone-Poulenc Rorer (now Sanofi), Indena SpA,

Figure 23. Tumor-targeted drug delivery system based on anasymmetric bowtie PAMAM dendrimer designed for the targeteddelivery of second-generation taxoid/fluorotaxoid bearing an imagingarm.



mailto:[email protected]

Mitsubishi Chemical Corp., Ajinomoto Co., Inc., Central GlassCo. Ltd., Japan Halon, Inc. (now Tohso F-Tech, Inc.), TokyoYuki Gosei Kogyo Co. Ltd., E. Merck, Darmstadt, and FujiChemical Ind. Co. Ltd. is acknowledged. The author alsothanks his research group members at the Sagami Institute, whoparticipated in the pioneering work on fluorine chemistry. He isgrateful to all of his former and current graduate students,postdoctoral research associates, senior staff assistants, and staffof the Institute of Chemical Biology & Drug Discovery(ICB&DD), especially those who were engaged in the “fluorinechemistry” projects at Stony Brook, for their cooperation,support, and hard work, which made my endeavor fruitful. Forthe cover art, the author thanks Dr. Raphael Geney fordesigning the background graphics, showing a tubulin-boundREDOR−Taxol molecule and a macrocyclic analogue, as wellas Alexandra A. Athan for technical assistance to put togetherthe images.

■ REFERENCES(1) Begue, J.-P.; Bonnet-Delpon, D. J. Fluorine Chem. 2006, 127, 992.(2) Isanbor, C.; O’Hagan, D. J. Fluorine Chem. 2006, 127, 303.(3) Ojima, I. Fluorine in Medicinal Chemistry and Chemical Biology;Wiley-Blackwell: Chichester, 2009.(4) Polina Cormier, E.; Das, M.; Ojima, I. In Fluorine in MedicinalChemistry and Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell:Chichester, 2009, p 525.(5) MedAdNews 2007, 13, 200. See also: http://business.highbeam.com/437048/article-1G1-167388389/med-ad-news-200-bestselling-prescription-medicinescompanies.(6) Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.(7) O’Hagan, D.; Schaffrath, C.; Cobb, S. L.; Hamilton, J. T. G.;Murphy, C. D. Nature 2002, 416, 279.(8) Martino, R.; Malet-Martino, M.; Gilard, V. Curr. Drug Metab.2000, 1, 271.(9) Wadhwani, P.; Strandberg, E. In Fluorine in Medicinal Chemistryand Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell: Chichester,2009, p 463.(10) Kilbourn, M. R.; Shao, X. In Fluorine in Medicinal Chemistry andChemical Biology; Ojima, I., Ed.; Wiley-Blackwell: Chichester, 2009, p361.(11) Uneyama, K. Organofluorine Chemistry; Blackwell: Oxford, 2006.(12) Soloshonok, V. A. Fluorine-Containing Synthons; ACSSymposium Series 911; American Chemical Society: Washington,D.C., 2005.(13) Soloshonok, V. A.; Mikami, K.; Yamazaki, T.; Welch, J. T.;Honek, J. F. Current Fluoroorganic Chemistry: New Synthetic Directions,Technologies, Materials, and Biological Applications; ACS SymposiumSeries 949; American Chemical Society: Washington, D.C., 2007; Vol.949.(14) Kirsch, P. Modern Fluoroorganic Chemistry: Synthesis, Reactivity,Applications; Wiley−VCH: Stuttgart, 2004.(15) Hiyama, T. Organofluorine Compounds: Chemistry andApplications; Springer−Verlag: Stuttgart, 2000.(16) Soloshonok, V. A. Enantiocontrolled Synthesis of FluoroorganicCompounds: Stereochemical Challenges and Biomedicinal Targets; Wiley:New York, 1999.(17) Kitazume, T.; Yamazaki, T. Experimental Methods in OrganicFluorine Chemistry; Kodansha, Gordon and Breach Science Publisher:Tokyo, 1998.(18) Hudlicky, M.; Pavlath, A. E. Chemistry of Organic FluorineCompounds II: A Critical Review; American Chemical Society:Washington, D.C., 1995.(19) Kukhar, V. P.; Soloshonok, V. A. Fluorine-Containing AminoAcids: Synthesis and Properties; Wiley: Chichester, 1994.(20) Clark, L.; Gollan, R. Science 1966, 152, 1755.

(21) Ojima, I.; Tsai, C. Y.; Tzamarioudaki, M.; Bonafoux, D. InOrganic Reactions; Overman, L. E., Ed.; Wiley: New York, 2000; Vol.56, p 1.(22) Pino, P.; Piacenti, F.; Bianchi, M. In Organic Syntheses via MetalCarbonyls; Wender, I., Pino, P., Ed.; Wiley-Interscience: New York,1977; Vol. 2, p 43.(23) Cornils, B. In New Syntheses with Carbon Monoxide; Falbe, J.,Ed.; Springer-Verlag: Berlin, 1980; p 1.(24) Ojima, I. Chem. Rev. 1988, 88, 1011.(25) Filler, R. Biochemistry Involving Carbon−Fluorine Bonds;American Chemical Society: Washington, D.C., 1976.(26) Filler, R. CHEMTECH 1973, 752.(27) Smith, F. A. CHEMTECH 1973, 422.(28) Filler, R., Kobayashi, Y. Biomedical Aspects of Fluorine Chemistry;Elsevier Biomedical: Amsterdam, 1982.(29) Fuchikami, T.; Ojima, I. J. Am. Chem. Soc. 1982, 104, 3527.(30) Ojima, I.; Kato, K.; Okabe, M.; Fuchikami, T. J. Am. Chem. Soc.1987, 109, 7714.(31) Ojima, I.; Fuchikami, T. U.S. Patent 4370504, 1983.(32) Pino, P.; Piacenti, F.; Bianchi, M.; Lazzaroni, R. Chim. Ind.(Milan) 1968, 50, 106.(33) Schwager, I.; Knifton, J. F. Ger. Offen. 2 322 751, 1973; Chem.Abstr. 1974, 80, 70327m.(34) Booth, B. L.; Else, M. J.; Fields, R.; Haszeldine, R. N. J.Organomet. Chem. 1971, 27, 119.(35) Ojima, I. Actual. Chimique 1987, 171.(36) Falbe, J. New Syntheses with Carbon Monoxide; Springer-Verlag:Berlin, 1980.(37) Fuchikami, T.; Ohishi, K.; Ojima, I. J. Org. Chem. 1983, 48,3803.(38) Welch, J. T. Tetrahedron 1987, 43, 3123.(39) Imperialli, B. In Advances in Biotechnological Processes; Mizrahi,A., Ed.; Alan R. Liss Inc.: New York, 1988; Vol. 10, p 97.(40) Imperialli, B.; Abeles, R. H. Biochemistry 1986, 26, 3760.(41) Lamden, L.; Bartlett, P. A. Biochem. Biophys. Res. Commun. 1983,112, 1085.(42) Thaisrivongs, S.; Pals, D. T.; Kati, W. M.; Turner, S. R.;Thomasco, L. M.; Watt, M. J. Med. Chem. 1986, 29, 2080.(43) Feuerstein, G.; Lozovsky, D.; Cohen, L. A.; Labroo, V. M.; Kirk,K.; Kopkin, I. J.; Faden, A. I. Neuropeptides 1984, 4, 303.(44) Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami, T.; Fujita, M. J.Org. Chem. 1989, 54, 4511.(45) Yamaguchi, S.; Mosher, H. S. J. Org. Chem. 1973, 38, 1870.(46) Walborsky, H. M.; Baum, M.; Loncrini, D. F. J. Am. Chem. Soc.1955, 77, 3637.(47) Meng, H.; Clark, C. A.; Kumar, K. In Fluorine in MedicinalChemistry and Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell:Chichester, 2009; p 411.(48) diPAMP = (1R,2R)-1,2-bis[(o-anisylphenyl)phosphino]ethane.See: Knowles, W. S.; Sabacky, M. J.; Vineyard, B. D.; Weinkauff, D. J. J.Am. Chem. Soc. 1976, 97, 2567.(49) Fujita, M.; Ojima, I. Tetrahedron Lett. 1983, 24, 4573.(50) Knorre, D. G.; Lavrik, O. I.; Petrova, T. D.; Savachenko, T. I.;Yakobson, G. G. FEBS Lett. 1971, 12, 204.(51) Nevinsky, G. A.; Favorova, O. O.; Lavrik, O. I.; Petrova, T. D.;Kochkina, L. L.; Savachenko, T. I. FEBS Lett. 1974, 43, 135.(52) Pless, S. A.; Galpin, J. D.; Niciforovic, A. P.; Ahern, C. A. NatureChem. Biol. 2011, 7, 617.(53) Budisa, N. Engineering the Genetic Code; Wiley-VCH: Weinheim,2006.(54) Henne, A. L.; Naer, M. J. J. Am. Chem. Soc. 1951, 73, 1042.(55) Fuchikami, T.; Yamanouchi, A.; Ojima, I. Synthesis 1984, 766.(56) Ito, H.; Miller, D. C.; Willson, C. G. Macromolecules 1982, 15,915.(57) Koishi, T.; Tanaka, I.; Yasumura, T.; Ojima, I.; Fuchikami, T.Jpn. Patent 1676867, 1992.(58) Ojima, I.; Fuchikami, T. Jpn. Patent 1418495, 1988.(59) Ojima, I.; Fuchikami, T. Jpn. Patent 1640707, 1992.



http://business.highbeam.com/437048/article-1G1-167388389/med-ad-news-200-bestselling-prescription-medicinescompanies



(60) Ito, H.; Truong, H. D.; Okazaki, M.; Miller, D. C.; Fender, N.;Brock, P. J.; Wallraff, G. M.; Larson, C. E.; Allen, R. D. J. Photopolym.Sci. Technol. 2002, 15, 591.(61) Shirai, M.; Takashiba, S.; Tsunooka, M. J. Photopolym. Sci.Technol. 2003, 16, 545.(62) Cracowski, J.-M.; Montembault, V.; Hardy, I.; Bosc, D.;Ameduri, B.; Fontaine, L. J. Polym. Sci. A - Polym. Chem. 2008, 46,4383.(63) Ojima, I.; Nakahashi, K. Japan Kokai Tokkyo Koho 1990, Hei 4,49298.(64) Ojima, I.; Nakahashi, K. U.S. Patent 5276137, 1994.(65) Schoenberg, A.; Heck, R. F. J. Org. Chem. 1974, 39, 3327.(66) Fuchikami, T.; Ojima, I. Tetrahedron Lett. 1982, 23, 4099.(67) Heidelberger, C.; Barsons, D. G.; Remy, D. C. J. Am. Chem. Soc.1962, 84, 3597.(68) Ojima, I.; Fuchikami, T. Jpn. Patent 1634611, 1992.(69) O’Brien, W.; Taylor, J. Invest. Ophthalmol. Vis. Sci. 1991, 32,2455.(70) Ojima, I.; Okabe, M.; Kato, K.; Kwon, H. B.; Horvlth, I. T. J.Am. Chem. Soc. 1988, 210, 150.(71) Horvath, I. T.; Bor, G.; Garland, M.; Pino, P. Organometallics1986, 5, 1441.(72) Bor, G. Pure Appl. Chem. 1986, 58, 543.(73) Spindler, F.; Bor, G.; Dietler, U.; Pino, P. J. Organomet. Chem.1981, 213, 303.(74) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A.Biochemistry 1977, 16, 5484.(75) Ondetti, M. A.; Rubin, B.; Cushman, D. W. Science 1977, 196,441.(76) Patchett, A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J.; Taub,D.; Peterson, E. R.; Ikeler, T. J.; ten Broeke, J.; Payne, L. G.; Ondeyka,D. L.; Thorsett, E. D.; Greenlee, W. J.; Lohr, N. S.; Hoffsommer, R.D.; Joshua, H.; Ruyle, W. V.; Rothrack, J. W.; Aster, S. D.; Maycock, A.L.; Robinson, F. M.; Hirschmann, R.; Sweet, C. S.; Ulm, E. H.; Gross,D. M.; Vassil, T. C.; Stone, C. A. Nature 1980, 288, 280.(77) Filler, R. J. Fluorine Chem. 1986, 33, 361.(78) Ojima, I.; Jameison, F. A.; Pete, B.; Radunz, H.; Schittenhelm,C.; Lindner, H. J.; Smith, A. Drug Design Discov. 1994, 11, 91.(79) Ojima, I.; Jameison, F. A. Bioorg. Med. Chem. Lett. 1991, 1, 581.(80) Holmquist, B.; Bunning, P.; Riordan, J. F. Anal. Biochem. 1979,95, 540.(81) Smith, A. E.; Lindner, H. J. J. Computer-Aided Mol. Design 1991,5, 235.(82) Hansen, P. E.; Morgan, B. A. In The Peptides − Analysis,Synthesis, Biology; Vol 6. Opioid Peptides: Biology, Chemistry, andGenetics; Undenfriend, S., Meienhofer, J., Eds.; Academic Press:Waltham, 1984; p 269.(83) Paterson, S. J.; Robson, L. E.; Kosterlitz, H. W. In The Peptides− Analysis, Synthesis, Biology; Vol 6. Opioid Peptides: Biology, Chemistry,and Genetics; Undenfriend, S., Meienhofer, J., Eds.; Academic Press:Waltham, 1984; p 147.(84) Watanabe, J.; Tokuyama, S.; Takahashi, M.; Kaneto, H.; Maeda,M.; Kawasaki, K.; Taguchi, T.; Kobayashi, Y.; Yamamoto, Y.;Shimokawa, K. J. Pharmacobio-Dyn. 1991, 14, 101.(85) Thorsett, E. D.; Wyvratt, M. J. In Neuropeptides and TheirPeptidases; Turner, A. J., Ed.; Ellis Horwood-VCH: Chichester, 1987; p229.(86) Turner, A. J. In Neuropeptides and Their Peptidases; Turner, A. J.,Ed.; Ellis Horwood-VCH: Chichester, 1987; p 183.(87) McKelvy, J. F. Annu. Rev. Neurosci. 1986, 9, 415.(88) Ojima, I.; Jameison, F. A.; Conway, J. D.; Nakahashi, K.;Hagiwara, M.; Miyamae, T.; Radunz, H. E. Bioorg. Med. Chem. Lett.1992, 2, 219.(89) Ojima, I. In Organofluorine Compounds in Medicinal Chemistryand Biomedical Applications; Filler, R., Kobayashi, Y., Yagupolskii, L.M., Eds.; Elsevier: Amsterdam, 1993, p 241.(90) Juaristi, E. Enantioselective Synthesis of β-Amino Acids: Wiley-VCH: New York, 1997.(91) Cole, D. C. Tetrahedron 1994, 50, 9517.

(92) Ojima, I.; Delaloge, F. In Peptidomimetics Protocols; Kamierski,W., Ed.; Humana Press: New York, 1998, p 137.(93) Kingston, D. G. I. Chem. Commun. 2001, 10, 867.(94) Ojima, I.; Lin, S.; Wang, T. Curr. Med. Chem. 1999, 6, 927.(95) Georg, G. I.; Chen, T. T.; Ojima, I.; Vyas, D. Taxane AnticancerAgents: Basic Science and Current Status; American Chemical Society:Washington D.C., 1995; Vol. 583.(96) Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M.; Takeuchi, T. J.Antibiot. 1976, 29, 97.(97) Pearson, W. H. H. J. Org. Chem. 1989, 54, 4235.(98) Roers, R.; Verdine, G. L. Tetrahedron Lett. 2001, 42, 3563.(99) Nagai, M.; Kojima, F.; Naganawa, H.; Hamada, M.; Aoyagi, T.;Takeuchi, T. J. Antibiot. 1997, 50, 82.(100) Okino, T.; Matsuda, H.; Murakami, M.; Yamaguchi, K.Tetrahedron Lett. 1993, 34, 501.(101) Mimoto, T.; Hattori, N.; Takaku, H.; Kisanuki, S.; Fukazawa,T.; Terashima, K.; Kato, R.; Nojima, S.; Misawa, S.; Ueno, T.; Imai, J.;Enomoto, H.; Tanaka, S.; Sakikawa, H.; Shintani, M.; Hayashi, H.;Kiso, Y. Chem. Pharm. Bull. 2000, 48, 1310.(102) Kiso, Y.; Matsumoto, S.; Mimoto, H.; Kato, T.; Nojima, R.;Takaku, S.; Fukazawa, H.; Kimura, T.; Akaji, T. Arch. Pharm. 1998, 87.(103) Ojima, I.; McCarthy, J. M.; Welch, J. T. Biomedical Frontiers ofFluorine Chemistry, ACS Symp. Series 639; American Chemical Society:Washington, D.C., 1996.(104) Ojima, I. ChemBioChem 2004, 5, 628.(105) Hook, D. F.; Gessier, F.; Noti, C.; Kast, P.; Seebach, D.ChemBioChem 2004, 5, 691.(106) Ojima, I.; Inoue, T.; Chakravarty, S. J. Fluorine Chem. 1999, 97,3.(107) Ojima, I.; Inoue, T.; Slater, J. C.; Lin, S.; Kuduk, S. C.;Chakravarty, S.; Walsh, J. J.; Gilchrist, L.; McDermott, A. E.; Cresteil,T.; Monsarrat, B.; Pera, P.; Bernacki, R. J. In Asymmetric FluoroorganicChemistry: Synthesis, Application, and Future Directions; ACSSymposium Series 746; Ramachandran, P. V., Ed.; American ChemicalSociety: Washington, D.C., 1999, p 158.(108) O’Hagan, D.; Schaffrath, C.; Cobb, S. L.; Hamilton, J. T. G.;Cormac, D.; Murphy, C. D. Nature 2002, 416, 279.(109) Gerhard, U.; Thomas, S.; Mortishire-Smith, R. J. Pharm. Biol.Anal. 2003, 32, 531.(110) Kaneko, S.; Yamazaki, T.; Kitazume, T. J. Org. Chem. 1993, 58,2302.(111) Abouabdellah, A.; Begue, J. P.; Bonnet-Delpon, D.; Nga, T. T.T. J. Org. Chem. 1997, 62, 8826.(112) Uneyama, K.; Hao, J.; Amii, H. Tetrahedron Lett. 1998, 39,4079.(113) Soloshonok, V. A.; Soloshonok, I. V.; Kukhar, V. P.; Svedas, V.K. J. Org. Chem. 1998, 63, 1878.(114) Fustero, S.; Pina, B.; Salavert, E.; Navarro, A.; Ramírez deArellano, M. C.; Simon Fuentes, A. J. Org. Chem. 2002, 67, 4667.(115) Ojima, I. Acc. Chem. Res. 1995, 28, 383.(116) Ojima, I.; Delaloge, F. Chem. Soc. Rev. 1997, 26, 377.(117) Deshmukh, A. R.; Bhawal, B. M.; Krishnaswamy, D.; Govande,V. V.; Shinkre, B. A.; Jayanthi, A. Curr. Med. Chem. 2004, 11, 1889.(118) Alcaide, B.; Almendros, P. Curr. Med. Chem. 2004, 11, 1921.(119) Palomo, C.; Aizpurua, J. M.; Ganboa, I.; Oiardide, M. Curr.Med. Chem. 2004, 11, 1837.(120) Kamath, A.; Ojima, I. Tetrahedron 2012, 68, 10640.(121) Brieva, R.; Crich, J. Z.; Sih, C. J. J. Org. Chem. 1993, 58, 1068.(122) Kuznetsova, L.; Ungureanu, I. M.; Pepe, A.; Zanardi, I.; Wu, X.;Ojima, I. J. Fluorine Chem. 2004, 125, 487.(123) Ojima, I.; Kuznetsova, L.; Ungureanu, I. M.; Pepe, A.; Zanardi,I.; Chen, J. In Fluorine-Containing Synthons; ACS Symposium Series911; Soloshonok, V. A., Ed.; American Chemical Society/OxfordUniversity Press: Washington, D.C., 2005, p 544.(124) Ojima, I.; Slater, J. C.; Pera, P.; Veith, J. M.; Abouabdellah, A.;Begue, J.-P.; Bernacki, R. J. Bioorg. Med. Chem. Lett. 1997, 7, 133.(125) Trost, B. M. Acc. Chem. Res. 2002, 35, 695.(126) Ojima, I.; Sun, C. M.; Park, Y. H. J. Org. Chem. 1994, 59, 1249.



(127) Ojima, I.; Wang, T.; Delaloge, F. Tetrahedron Lett. 1998, 39,3663.(128) Ojima, I.; Wang, T.; Ng, E. W. Tetrahedron Lett. 1998, 39, 923.(129) Rowinsky, E. K. Annu. Rev. Med. 1997, 48, 353.(130) Bristol-Myers Squibb, 2003, http://packageinserts.bms.com/pi/pi_taxol.pdf.(131) National Cancer Institute, 2012, http://www.cancer.gov/cancertopics/druginfo/docetaxel.(132) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665.(133) Jordan, M. A.; Toso, R. J.; Wilson, L. Proc. Natl. Acad. Sci.U.S.A. 1993, 90, 9552.(134) Ojima, I.; Kuduk, S. D.; Chakravarty, S.; Ourevitch, M.; Begue,J.-P. J. Am. Chem. Soc. 1997, 119, 5519.(135) Georg, G. I.; Harriman, G. C. B.; Vander Velde, D. G.; Boge, T.C.; Cheruvallath, Z. S.; Datta, A.; Hepperle, M.; Park, H.; Himes, R.H.; Jayasinghe, L. In Taxane Anticancer Agents: Basic Science andCurrent Status; ACS Symposium Series 583; Georg, G. I., Chen, T. T.,Ojima, I., Vyas, D. M., Eds.; American Chemical Society: Washington,D.C., 1995; p 217.(136) Gueritte-Voegelein, F.; Mangatal, L.; Guenard, D.; Potier, P.;Guilhem, J.; Cesario, M.; Pascard, C. Acta Crstallogr. 1990, C46, 781.(137) Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.;Chirlian, L. E.; Francl, M. M.; Heerding, J. M.; Krauss, N. E. Can. J.Chem. 1994, 252.(138) Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.;Chirlian, L. E.; Francl, M. M.; Heerding, J. M.; Krauss, N. E.Tetrahedron 1993, 49, 6545.(139) Vander Velde, D. G.; Georg, G. I.; Grunewald, G. L.; Gunn, C.W.; Mitscher, L. A. J. Am. Chem. Soc. 1993, 115, 11650.(140) Mastropaolo, D.; Camerman, A.; Luo, Y.; Brayer, G. D.;Camerman, N. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6920.(141) Paloma, L. G.; Guy, R. K.; Wrasidlo, W.; Nicolaou, K. C. Chem.Biol. 1994, 2, 107.(142) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002,2, 48.(143) Ojima, I.; Slater, J. C.; Michaud, E.; Kuduk, S. D.; Bounaud, P.-Y.; Vrignaud, P.; Bissery, M.-C.; Veith, J.; Pera, P.; Bernacki, R. J. J.Med. Chem. 1996, 39, 3889.(144) Ojima, I.; Chen, J.; Sun, L.; Borella, C.; Wang, T.; Miller, M.;Lin, S.; Geng, X.; Kuznetsova, L.; Qu, C.; Gallagher, G.; Zhao, X.;Zanardi, I.; Xia, S.; Horwitz, S.; Clair, J. S.; Guerriero, J.; Bar-Sagi, D.;Veith, J.; Pera, P.; Bernacki, R. J. Med. Chem. 2008, 51, 3203.(145) Ojima, I.; Wang, T.; Miller, M. L.; Lin, S.; Borella, C. P.; Geng,X.; Pera, P.; Bernacki, R. J. Bioorg. Med. Chem. Lett. 1999, 9, 3423.(146) Ojima, I.; Kuduk, S.; Slater, J.; Gimi, R.; Sun, C.-M.Tetrahedron 1996, 52, 209.(147) Kuznetsova, L. V.; Pepe, A.; Ungureanu, I. M.; Pera, P.;Bernacki, R. J.; Ojima, I. J. Fluorine Chem. 2008, 129, 817.(148) Ojima, I.; Slater, J. C. Chirality 1997, 9, 487.(149) Gut, I.; Ojima, I.; Vaclavikova, R.; Simek, P.; Horsky, S.;Linhart, I.; Soucek, P.; Knodrova, E.; Kuzetsova, L.; Chen, J.Xenobiotica 2006, 36, 772.(150) Vuilhorgne, M.; Gaillard, C.; Sanderlink, G. J.; Royer, I.;Monsarrat, B.; Dubois, J.; Wright, M. In Taxane Anticancer Agents:Basic Science and Current Status; ACS Symposium Series 583; Georg,G. I., Chen, T. T., Ojima, I., Vyas, D. M., Eds.; American ChemicalSociety: Washington, D.C., 1995; p 98.(151) Kuznetsova, L.; Sun, L.; Chen, J.; Zhao, X.; Seitz, J.; Das, M.;Li, Y.; Veith, J.; Pera, P.; Bernacki, R.; Xia, S.; Horwitz, S.; Ojima, I. J.Fluorine Chem. 2012, 143, 177.(152) Pepe, A.; Kuznetsova, L.; Sun, L.; Ojima, I. In Fluorine inMedicinal Chemistry and Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell: Chichester, 2009; p 117.(153) Li, Y.; Poliks, B.; Cegelski, L.; Poliks, M.; Gryczynski, Z.;Piszcek, G.; Jagtap, P. G.; Studelska, D. R.; Kingston, D. G. I.; Schaefer,J.; Bane, S. Biochemistry 2000, 39, 281.(154) Geney, R.; Sun, L.; Pera, P.; Bernacki Ralph, J.; Xia, S.; HorwitzSusan, B.; Simmerling Carlos, L.; Ojima, I. Chem. Biol. 2005, 12, 339.

(155) Rao, S.; He, L.; Chakravarty, S.; Ojima, I.; Orr, G. A.; Horwitz,S. B. J. Biol. Chem. 1999, 274, 37990.(156) Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998, 391,199.(157) Paik, Y.; Yang, C.; Metaferia, B.; Tang, S.; Bane, S.; Ravindra,R.; Shanker, N.; Alcaraz, A. A.; Johnson, S. A.; Schaefer, J.; O’Connor,R. D.; Cegelski, L.; Snyder, J. P.; Kingston, D. G. I. J. Am. Chem. Soc.2007, 129, 361.(158) Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. J. Mol. Biol. 2001,313, 1045.(159) Sun, L.; Simmerling, C.; Ojima, I. ChemMedChem 2009, 4, 719.(160) Ojima, I.; Kuznetsova, L. V.; Sun, L. In Current FluoroorganicChemistry. New Synthetic Directions, Technologies, Materials andBiological Applications; ACS Symposium Series 949; Soloshonok, V.,Mikami, K., Yamazaki, T., Welch, J. T., Honek, J., Eds.; AmericanChemical Society/Oxford University Press: Washington, DC, 2007; p288.(161) Jaracz, S.; Chen, J.; Kuznetsova, L.; Ojima, I. Bioorg. Med.Chem. 2005, 13, 5043.(162) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.;Kantoff, P. W.; Richie, J. P.; Langer, R. Proc. Natl. Acad. Sci. U.S.A.2006, 103, 6315.(163) Chu, T. C.; Marks, J. W., III; Lavery, L. A.; Faulkner, S.;Rosenblum, M. G.; Ellington, A. D.; Matthew Levy, M. Cancer Res.2006, 66, 5989.(164) Ducry, L.; Stump, B. Bioconjugate Chem 2010, 21, 5.(165) Xia, W.; Low, P. J. Med. Chem. 2010, 53, 6811.(166) Ojima, I.; Zuniga, E.; Berger, W.; Seitz, J. Future Med. Chem.2012, 4, 33.(167) Cobb, S.; Murphy, C. J. Fluorine Chem. 2009, 130, 132.(168) Reivich, M.; Kuhl, D.; Wolf, A.; Greenberg, J.; Phelps, M.; Ido,T.; Casella, V.; Fowler, J.; Hoffman, E.; Alavi, A.; Som, P.; Sokoloff, L.Circ. Res. 1979, 44, 127.(169) Ametamey, S.; Honer, M.; Schubiger, P. Chem. Rev. 2008, 108,1501.(170) Kimura, Y.; Simeon, F.; HAtazawa, J.; Mozley, P.; Pike, V.;Innis, R.; Fugita, M. Eur. J. Nucl. Med. Mol. I 2010, 37, 1943.(171) Meyer, B.; Mann, N.; Lewis, J.; Milligan, G.; Sinclair, A.; Howe,P. Lipids 2003, 38, 391.(172) Sauer, L.; Dauchy, R.; Blask, D. Cancer Res. 2000, 60, 5289.(173) Grammatikos, S.; Subbaiah, P.; Victor, T.; Miller, W. Br. J.Cancer 1994, 70, 219.(174) Bradley, M.; Webb, N.; Anthony, F.; Devanesan, P.; Wittman,P.; Hemamalini, S.; Chander, M.; Baker, S.; He, L.; Horowitz, S.;Swindell, C. Clin. Cancer Res. 2001, 7, 3229.(175) Seitz, J.; Ojima, I. In Drug Delivery in Oncology - From ResearchConcepts to Cancer Therapy; Kratz, F., Senter, P., Steinhagen, H., Eds.;Wiley-VCH: Weinheim, 2011; Vol. 3, p 1323.(176) Sparreboom, A.; Wolff, A.; Verweij, J.; Zabelina, Y.; vanZomeren, D. M.; McIntire, G.; Swindell, C.; Donehower, R.; Baker, S.Clin. Cancer Res. 2003, 9, 151.(177) Kuznetsova, L.; Chen, J.; Sun, X.; Wu, A.; Pepe, J.; Veith, P.;Pera, R.; Bernacki, R.; Ojima, I. Bioorg. Med. Chem. Lett. 2006, 16, 974.(178) Chen, J.; Jaracz, S.; Zhao, X.; Chen, S.; Ojima, I. Expert Opin.Drug Deliv. 2005, 2, 873.(179) Chari, R. V. J. Adv. Drug Delivery Rev. 1998, 31, 89.(180) Doronina, S. O.; Mendelsohn, B. A.; Bovee, T. D.; Cerveny, C.G.; Alley, S. C.; Meyer, D. L.; Oflazoghu, E.; Toki, B. E.; Sanderson, R.J.; Zabinski, R. F.; Wahl, A. F.; Senter, P. D. Bioconjugate Chem. 2006,17, 114.(181) Beck, A.; Haeuw, J. F.; Wurch, T.; Goetsch, L.; Bailly, C.;Corvaia, N. Discov. Med. 2010, 10, 329.(182) Ojima, I.; Geng, X.; Wu, X.; Qu, C.; Borella, C.; Xie, H.;Wilhelm, S.; Leece, B.; Bartle, L.; Goldmacher, V.; Chari, R. J. Med.Chem. 2002, 45, 5620.(183) Kigawa, J.; Minagawa, Y.; Kanamori, Y.; Itamochi, H.; Cheng,X.; Okada, M.; Oisho, T.; Terakawa, N. Cancer 1998, 82, 697.(184) Chen, J.; Chen, S.; Zhao, X.; Kuznetsova, L.; Wong, S.; Ojima,I. J. Am. Chem. Soc. 2008, 130, 16778.



http://packageinserts.bms.com/pi/pi_taxol.pdf

http://packageinserts.bms.com/pi/pi_taxol.pdf

http://www.cancer.gov/cancertopics/druginfo/docetaxel

http://www.cancer.gov/cancertopics/druginfo/docetaxel

(185) Ojima, I. Acc. Chem. Res. 2008, 41, 108.(186) Chen, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Wong,S.; Ojima, I. Bioconjugate Chem. 2010, 21, 979.(187) Russell-Jones, G.; McTavish, K.; McEwan, J.; Rice, J.;Nowotnik, D. J. Inorg. Biochem. 2004, 98, 1625.(188) Leamon, C. P.; Reddy, J. A. Adv. Drug Delivery Rev. 2004, 56,1127.(189) Zempleni, J. Ann. Rev. Nutr. 2005, 25, 175.(190) Zempleni, J.; Wijeratne, S.; Hassan, Y. Biofactors 2009, 35, 36.(191) Bianco, A.; Kostarelos, K.; Prato, M. Curr. Opin. Chem. Biol.2005, 9, 674.(192) Prato, M.; Kostarelos, K.; Bianco, A. Acc. Chem. Res. 2008, 41,60.(193) Gillies, E.; Frechet, J. Drug Discov. Today 2005, 10, 35.(194) Gaertner, H.; Cerini, F.; Kamath, A.; Rochat, A.-F.; Siegrist, C.-A.; Menin, L.; Hartley, O. Bioconjugate Chem. 2011, 22, 1103.



Documents

JO - Stony Brook University · 1. EXPLORATION OF ORGANOFLUORINE CHEMISTRY BY MEANS OF TRANSITION-METAL CATALYSIS My ﬁrst encounter with “ﬂuorine chemistry” was in the late